Multimeric VLA-4 antagonists comprising polymer moieties

ABSTRACT

Disclosed are conjugates which bind VLA-4. Certain of these conjugates also inhibit leukocyte adhesion and, in particular, leukocyte adhesion mediated by VLA-4. Such conjugates are useful in the treatment of inflammatory diseases in a mammalian patient, e.g., human, such as asthma, Alzheimer&#39;s disease, atherosclerosis, AIDS dementia, diabetes, inflammatory bowel disease, rheumatoid arthritis, tissue transplantation, tumor metastasis and myocardial ischemia. The conjugates can also be administered for the treatment of inflammatory brain diseases such as multiple sclerosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/586,975 filed Jul. 8, 2004, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to conjugates which inhibit leukocyte adhesionand, in particular, leukocyte adhesion mediated at least in part byVLA-4. The conjugates of this invention are characterized as containingmore than one VLA-4 inhibiting compound covalently attached to abio-compatible polymer, such as polyethylene glycol.

2. State of the Art

The physical interaction of inflammatory leukocytes with each other andother cells of the body plays an important role in regulating immune andinflammatory responses [Springer, T. A. Nature, 346, 425, (1990);Springer, T. A. Cell 76, 301, (1994)]. Many of these interactions aremediated by specific cell surface molecules collectively referred to ascell adhesion molecules. These adhesion molecules have been sub-dividedinto different groups on the basis of their structure. One family ofadhesion molecules which is believed to play a important role inregulating immune and inflammatory responses is the integrin family.This family of cell surface glycoproteins has a typical non-covalentlylinked heterodimer structure.

The particular integrin subgroup of interest herein involves the alpha 4(α₄) chain, which can pair with two different beta chains beta1 (β1) andbeta7 (β7) [Sonnenberg, A. ibid]. The α4β1 pairing occurs on manycirculating leukocytes (for example lymphocytes, monocytes andeosinophils) although it is absent or only present at low levels oncirculating neutrophils. VLA-4 (Very Late Antigen-4, also referred to asα₄β₁ integrin and as CD49d/CD29), first identified by Hemler and Takada¹is a member of the β1 integrin family of cell surface receptors. VLA-4consists of an α1 chain and a β1 chain. There are at least nine β1integrins, all sharing the same β1 chain and each having a distinct achain. These nine receptors all bind a different complement of thevarious cell matrix molecules, such as fibronectin, laminin, andcollagen. VLA-4, for example, binds to fibronectin. VLA-4 also bindsnon-matrix molecules that are expressed by endothelial and other cells.

VLA-4 (α4β1 integrin) binds to an adhesion molecule called Vascular CellAdhesion Molecule-1 (or VCAM-1) which is frequently up-regulated onendothelial cells at sites of inflammation [Osborne, L. Cell, 62, 3(1990)]. VCAM-1 is a non-matrix molecule which is an expressed receptorthat is believed to be responsible for trafficking leukocytes into thecentral nervous system (CNS). α_(4β)1 has also been shown to bind to atleast three sites in the matrix molecule fibronectin [Humphries, M. J.et al. Ciba Foundation Symposium, 189, 177, (1995)]. Distinct epitopesof VLA-4 are responsible for the fibronectin and VCAM-1 bindingactivities and each has been demonstrated to be independentlyinhibited.² Based on data obtained with monoclonal antibodies in animalmodels it is believed that the interaction between α4β1 and ligands onother cells and the extracellular matrix plays an important role inleukocyte migration and activation [Yednock, T. A. et al, Nature, 356,63, (1992).

The integrin generated by the pairing of α₄ and β7 has been termedLPAM-1 [Holzmann, B and Weissman, I. EMBO J. 8, 1735, (1989)] and likeα4β1, can bind to VCAM-1 and fibronectin. In addition, .alpha.4.beta.7binds to an adhesion molecule believed to be involved in the homing ofleukocytes to mucosal tissue termed MAdCAM-1 [Berlin, C. et al. Cell,74,185, (1993)]. The interaction between .α4β7 and MAdCAM-1 may also beimportant at sites of inflammation outside of mucosal tissue [Yang, X-D.et al. PNAS, 91,12604 (1994)].

Intercellular adhesion mediated by VLA-4 and other cell surfacereceptors is associated with a number of inflammatory responses. At thesite of an injury or other inflammatory stimuli, activated vascularendothelial cells express molecules that are adhesive for leukocytes.The mechanics of leukocyte adhesion to endothelial cells involve, inpart, the recognition and binding of cell surface receptors onleukocytes to the corresponding cell surface molecules on endothelialcells. Once bound, the leukocytes migrate across the blood vessel wallto enter the injured site and release chemical mediators to combatinfection. For reviews of adhesion receptors of the immune system, see,for example, Springer³ and Osborn⁴.

Inflammatory brain disorders, such as multiple sclerosis (MS),meningitis, encephalitis, and a disease model called experimentalautoimmune encephalomyelitis (EAE), are examples of central nervoussystem disorders in which the endothelium/leukocyte adhesion mechanismresults in destruction to otherwise healthy brain tissue. Large numbersof leukocytes migrate across the blood brain barrier (BBB) in subjectswith these inflammatory diseases. The leukocytes release toxic mediatorsthat cause extensive cell damage and death resulting in impaired nerveconduction and paralysis. Similar occurrences in encephalitis andmeningitis indicate that these diseases can be treated with suitablecell adhesion inhibitors.

In other organ systems, tissue damage also occurs via an adhesionmechanism resulting in migration or activation of leukocytes. Forexample, inflammatory bowel disease¹⁵ (including ulcerative colitis andCrohn's disease), are at least partially caused by leukocyte traffickingacross the intestinal endothelium via an α4β7 interaction with MadCAMand possibly α4β1 interaction with VCAM-1 expressed in this tissue aswell. Asthma⁶⁻⁸, rheumatoid arthritis¹⁸⁻²¹ and tissue transplantrejection²² are all thought to have components based in interaction ofα4β1 with VCAM-1 and/or fibronectin, probably both. it has been shownthat the initial insult following myocardial (heart tissue) ischemia canbe further complicated by leukocyte entry to the injured tissue causingstill further injury (Vedder et al.⁵). Other inflammatory or medicalconditions mediated by an adhesion molecule mechanism include, by way ofexample, Alzheimer's disease, atherosclerosis⁹⁻¹⁰, AIDS dementia¹¹,diabetes¹²⁻¹⁴ (including acute juvenile onset diabetes, tumormetastasis²³⁻²⁸, stroke, and other cerebral traumas, nephritis,retinitis, atopic dermatitis, psoriasis, and acute leukocyte-mediatedlung injury such as that which occurs in adult respiratory distresssyndrome.

One group of VLA-4 antagonists showing promise as anti-inflammatoryagents is the class of sulfonylated-Pro-Phe compounds as set forth in,for example, U.S. Pat. No. 6,489,300.³¹ These compounds are very potentantagonists of VLA-4/VCAM-1 binding.

Owing to extensive first pass liver metabolism, these compounds arepoorly orally available. Because many of the disease conditionstreatable by these compounds are chronic conditions, a prolonged serumhalf-life for the administered compound would increase the usefulness ofthese kinds of compounds in treating disease in mammals.

The half-life of a drug is a measure of the time that it takes for theamount of drug in the body to decrease by one half, through normalmetabolic and elimination pathways. VLA-4 inhibitors, including thosedisclosed in U.S. Pat. No. 6,489,300, suffer from short half-lives ofaround 10 to 20 minutes, even when intravenously administered in apharmaceutical formulation. In order for the patient to retain aneffective amount of the drug in their system for a reasonable period oftime, either very large quantities of the drug must be administeredand/or the drug must be administered many times in a day.

VLA-4 inhibitors with such short half-lives are not commercially viabletherapeutic candidates. Therefore, there is a need for VLA-4 inhibitorswith significantly enhanced serum half-lives; preferably in the range ofhours to days.

SUMMARY OF THE INVENTION

This invention provides conjugates exhibiting VLA-4 antagonisticproperties. The conjugates of this invention are characterized ascontaining more than one VLA-4 antagonists covalently attached to apolymer. Without being limited to any theory, the improved serumhalf-life is believed to be associated with covalent conjugation ofactive compounds to a polymer. In addition, attachment of multiplecopies of such compounds to the polymer minimizes degradation ofbiological efficacy.

In one aspect, the invention provides conjugates of formula I below:

-   B is a bio-compatible polymer moiety optionally covalently attached    to a branched-arm hub molecule;-   q is from about 2 to about 20;-   A at each occurrence is independently a compound of formula II    or a pharmaceutically acceptable salt thereof, wherein-   J is selected from:    -   a) a group of formula (a):    -   wherein R³¹ is a covalent bond to the polymer moiety which        optionally comprises a linker, or R³¹ is selected from the group        consisting of —H, R^(31′), —NH₂, —NHR^(31′), —N(R³¹′)₂,        —NC₃—C₆cyclic, —OR^(31′), and —SR^(31′), wherein each R^(31′) is        independently an optionally substituted straight or branched        C₁-C₆alkyl, optionally substituted C₃-C₆cycloalkyl, optionally        substituted aryl, or optionally substituted heteroaryl,    -   and R³² is a covalent bond to the polymer moiety which        optionally comprises a linker, or R³² is selected from the group        consisting of —H, —NO₂, haloalkyl, and —N(MR⁴¹)R⁴² wherein M is        a covalent bond, —C(O)— or —SO₂—, and R⁴¹ is R^(41′),        N(R^(41′))₂, or —OR^(41′),    -   wherein each R^(41′) is independently hydrogen, an optionally        substituted straight or branched C₁-C₆alkyl, optionally        substituted cycloalkyl, optionally substituted aryl, optionally        substituted heterocyclic or an optionally substituted        heteroaryl, wherein optional substitutions are halide,        C₁-C₆alkyl, or —OC₁-C₆alkyl,    -   and R⁴² is hydrogen, R^(41′), alkynyl, or substituted alkynyl;        and    -   b) a group of formula (b):    -   wherein R is selected from the group consisting of a covalent        bond to the polymer moiety, hydrogen, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, amino, hydroxyl, substituted        amino, alkyl, substituted alkyl, alkyloxy, aryloxy,        heteroaryloxy, heterocyclyloxy, thiol, arylthio, heteroarylthio,        heterocyclylthio and substituted alkyl wherein each amino,        substituted amino, alkyl, substituted alkyl, aryl, substituted        aryl, heteroaryl, and substituted heteroaryl is optionally        covalently bound to the polymer moiety wherein, in each case,        the polymer moiety optionally comprises a linker which        covalently links the polymer moiety;    -   Ar¹ is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl wherein each of        aryl, substituted aryl, heteroaryl and substituted heteroaryl is        optionally covalently bound to the polymer moiety wherein the        polymer moiety optionally comprises a linker which covalently        links the polymer moiety to Ar¹;    -   Ar² is selected from the group consisting of aryl, substituted        aryl, heteroaryl, substituted heteroaryl, alkyl, substituted        alkyl, alkylamino, and substituted alkylamino, wherein Ar² is        optionally covalently bound to the polymer moiety and wherein        the polymer moiety optionally comprises a linker which        covalently links the polymer moiety to Ar²;    -   X is selected from the group consisting of —NR¹—, —O—, —S—,        —SO—, —SO₂ and optionally substituted —CH₂— which is optionally        covalently bound to the polymer moiety wherein, in each case,        the polymer moiety optionally comprises a linker which        covalently links the polymer moiety;    -   where R¹ is selected from the group consisting of hydrogen and        alkyl;-   T is selected from:    -   a) a group of formula (c)    -   wherein Y is selected from the group consisting of —O— and —NR¹—        wherein R¹ is selected from the group consisting of hydrogen and        alkyl;    -   W is selected from the group consisting of a covalent bond to a        polymer moiety which optionally comprises a linker and —NR²R³        wherein R² and R³ are independently selected from the group        consisting of hydrogen, alkyl, substituted alkyl, and where R²        and R³, together with the nitrogen atom bound thereto, form a        heterocyclic ring or a substituted heterocyclic ring wherein        each of alkyl, substituted alkyl, heterocyclic and substituted        heterocyclic is optionally covalently bound to a polymer moiety        which further optionally comprises a linker;    -   m is an integer equal to 0, 1 or 2;    -   n is an integer equal to 0, 1 or 2; and    -   b) a group of formula (d)    -   wherein G is an optionally substituted aryl or optionally        substituted heteroaryl 5 or 6 membered ring containing 0 to 3        nitrogens, wherein said aryl or heteroary optionally further        comprises a covalent bond to a polymer moiety which optionally        comprises a linker;    -   R⁶ is a covalent bond to a polymer moiety which optionally        comprises a linker, or R⁶ is —H, alkyl, substituted alkyl, or        —CH₂C(O)R¹⁷, wherein R¹⁷ is —OH, —OR¹⁸, or —NHR¹⁸, wherein R¹⁸        is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,        or substituted heteroaryl;-   R⁵⁵ is —OH or a hydrolyzable ester, or R⁵⁵ forms a hydrolyzable    polymer ester with the polymer moiety, optionally through a linker;    provided that:    -   A. at least one of Ar¹, J, Ar², R⁵⁵ and T (more preferably one        of J and T) contains a covalent bond to the polymer moiety;    -   B. when X is —O—, then m is two; and    -   C. the conjugate of formula I has a molecular weight of no more        than about 80,000.

The invention also provides pharmaceutical compositions whichcompositions comprise, for example, a pharmaceutically acceptablecarrier and a therapeutically effective amount of a conjugate of theinvention or mixtures thereof.

The invention also provides methods for treating a disease mediated, atleast in part, by VLA-4 in a patient, which method comprisesadministering a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a therapeutically effective amount of a conjugateof the invention or mixtures thereof.

The invention also includes the use of a conjugate of the invention, andpharmaceutically acceptable salts thereof, for the manufacture of amedicament for use in treating a disease mediated, at least in part, byVLA-4 in a patient.

The conjugates and pharmaceutical compositions may be used to treatdisease conditions mediated, at least in part, by VLA-4 or leukocyteadhesion. Such disease conditions include, by way of example, asthma,Alzheimer's disease, atherosclerosis, AIDS dementia, diabetes (includingacute juvenile onset diabetes), inflammatory bowel disease (includingulcerative colitis and Crohn's disease), multiple sclerosis, rheumatoidarthritis, tissue transplantation, tumor metastasis, meningitis,encephalitis, stroke, and other cerebral traumas, nephritis, retinitis,Sjogren's disease, atopic dermatitis, psoriasis, myocardial ischemia andacute leukocyte-mediated lung injury such as that which occurs in adultrespiratory distress syndrome.

Other disease conditions which may be treated using conjugates andcompositions of the present invention include, but are not limited to,inflammatory conditions such as erythema nodosum, allergicconjunctivitis, optic neuritis, uveitis, allergic rhinitis, ankylosingspondylitis, psoriatic arthritis, vasculitis, Reiter's syndrome,systemic lupus erythematosus, progressive systemic sclerosis,polymyositis, dermatomyositis, Wegner's granulomatosis, aortitis,sarcoidosis, lymphocytopenia, temporal arteritis, pericarditis,myocarditis, congestive heart failure, polyarteritis nodosa,hypersensitivity syndromes, allergy, hypereosinophilic syndromes,Churg-Strauss syndrome, chronic obstructive pulmonary disease,hypersensitivity pneumonitis, chronic active hepatitis, interstitialcystitis, autoimmune endocrine failure, primary biliary cirrhosis,autoimmune aplastic anemia, chronic persistent hepatitis andthyroiditis.

Preferably, the conjugates and pharmaceutically compositions of thisinvention are used in methods for treating asthma, rheumatoid arthritisand multiple sclerosis. As to this latter disease, the conjugates ofthis invention not only provide an anti-inflammatory effect whenadministered in vivo but further find use in treating conditions anddiseases associated with demyelination.

The invention also provides methods of preparing the conjugates of theinvention and the intermediates used in those methods.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, this invention relates to conjugates which inhibitleukocyte adhesion and, in particular, leukocyte adhesion mediated, atleast in part, by VLA-4.

In one preferred embodiment, only one of Ar¹, J, Ar², and T contains acovalent bond to a polymer moiety.

In another preferred embodiment, the polymer moiety is attached to the—NR²R³ group.

In a further preferred embodiment, when X in the conjugates of theinvention is NR¹, then m is two.

In yet another preferred embodiment, q is an integer of from 2 to about20 and more preferably from 2 to about 8.

Preferred conjugates of formula I include those of formula Ia below:

and pharmaceutically acceptable salts thereof, wherein

-   B is a a di-valent, tri-valent, tetra-valent or higher valency    bio-compatible polymer moiety or optionally more than one    biocompatible polymers covalently joined by a functional group    linkage or by a branched-arm hub molecule or both to form a    di-valent, tri-valent, tetra-valent or higher valency polymer    moiety;-   q is from 2 to about 20;-   A at each occurrence is independently a compound of formula IIa    wherein-   R is selected from the group consisting of a covalent bond to the    polymer moiety, amino, substituted amino, alkyl and substituted    alkyl wherein each amino, substituted amino, alkyl and substituted    alkyl is optionally covalently bound to the polymer moiety wherein,    in each case, the polymer moiety optionally comprises a linker which    covalently links the polymer moiety;-   Ar¹ is selected from the group consisting of aryl, substituted aryl,    heteroaryl and substituted heteroaryl;-   Ar² is selected from the group consisting of aryl, substituted aryl,    heteroaryl and substituted heteroaryl wherein each of aryl,    substituted aryl, heteroaryl and substituted heteroaryl is    optionally covalently bound to the polymer moiety wherein the    polymer moiety optionally comprises a linker which covalently links    the polymer moiety to Ar²;-   X is selected from the group consisting of —NR¹—, —O—, —S—, —SO—,    —SO₂ and optionally substituted —CH₂— where R¹ is selected from the    group consisting of hydrogen and alkyl;-   Y is selected from the group consisting of —O— and —NR¹— wherein R¹    is selected from the group consisting of hydrogen and alkyl;-   W is selected from the group consisting of a covalent bond to the    polymer moiety which optionally comprises a linker and —NR²R³    wherein R² and R³ are independently selected from the group    consisting of hydrogen, alkyl, substituted alkyl, and where R² and    R³, together with the nitrogen atom bound thereto, form a    heterocyclic ring or a substituted heterocyclic ring wherein each of    alkyl, substituted alkyl, heterocyclic and substituted heterocyclic    is optionally covalently bound to the polymer moiety optionally    through a linker;-   m is an integer equal to 0, 1 or 2;-   n is an integer equal to 0, 1 or 2; and    pharmaceutically acceptable salts thereof;    provided that:    -   A. at least one of R, Ar², W and —NR²R³ contain a covalent bond        to the polymer moiety;    -   B. when R is covalently bound to the polymer moiety, n is one        and X is not —O—, —S—, —SO—, or —SO₂—;    -   C. when X is —O— or —NR¹—, then m is two; and        D. the conjugate of formula Ia has a molecular weight of no more        than 60,000.

Preferred conjugates of formula I include those of formula Ib below:

-   -   wherein each A is independently a compound of formula IIb below:    -   and wherein q is 2 to about 20;    -   B is as defined above;    -   Ar¹ is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl;    -   Ar² is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl wherein each of        aryl, substituted aryl, heteroaryl and substituted heteroaryl is        optionally covalently bound to a polymer moiety wherein the        polymer moiety optionally comprises a linker which covalently        links the polymer moiety to Ar²;    -   Y is selected from the group consisting of —O— and —NR¹— wherein        R¹ is selected from the group consisting of hydrogen and alkyl;    -   W is selected from the group consisting of a covalent bond to a        polymer moiety which optionally comprises a linker and —NR²R³        wherein R² and R³ are independently selected from the group        consisting of hydrogen, alkyl, substituted alkyl, and where R²        and R³, together with the nitrogen atom bound thereto, form a        heterocyclic ring or a substituted heterocyclic ring wherein        each of alkyl, substituted alkyl, heterocyclic and substituted        heterocyclic is optionally covalently bound to the polymer        moiety which further optionally comprises a linker;    -   provided that at least one of Ar², W and —NR²R³ is covalently        bound to a polymer moiety which optionally comprises a linker;    -   and further provided that the conjugate of formula Ib has a        molecular weight of no more than 60,000.

Preferred conjugates of formula I include those of formula Ic below:

-   -   wherein each A is independently a compound of formula IIc below:    -   and wherein q is 2 to about 20;    -   B is as defined above;    -   R is selected from the group consisting of a covalent bond to a        polymer moiety, amino, substituted amino, alkyl and substituted        alkyl wherein each amino, substituted amino, alkyl and        substituted alkyl is optionally covalently bound to the polymer        moiety wherein, in each case, the polymer moiety optionally        comprises a linker which covalently links the polymer moiety;    -   Ar¹ is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl;    -   Ar² is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl wherein each of        aryl, substituted aryl, heteroaryl and substituted heteroaryl is        optionally covalently bound to a polymer moiety wherein the        polymer moiety optionally comprises a linker which covalently        links the polymer moiety to Ar²;    -   Y is selected from the group consisting of —O— and —NR¹— wherein        R¹ is selected from the group consisting of hydrogen and alkyl;    -   W is selected from the group consisting of a covalent bond to a        polymer moiety which optionally comprises a linker and —NR²R³        wherein R² and R³ are independently selected from the group        consisting of hydrogen, alkyl, substituted alkyl, and where R²        and R³, together with the nitrogen atom bound thereto, form a        heterocyclic ring or a substituted heterocyclic ring wherein        each of alkyl, substituted alkyl, heterocyclic and substituted        heterocyclic is optionally covalently bound to a polymer moiety        which further optionally comprises a linker;    -   n is an integer equal to 0, 1 or 2; and    -   pharmaceutically acceptable salts thereof;    -   provided that at least one of R, Ar², W and —NR²R³ is covalently        bound to a polymer moiety which optionally comprises a linker;    -   and further provided that the conjugate of formula Ic has a        molecular weight of no more than 60,000.

Preferred conjugates of formula I include those of formula Id below:

-   -   wherein each A is independently a compound of formula IId below:    -   and wherein q is 2 to about 20;    -   B is as defined above;    -   R is selected from the group consisting of a covalent bond to a        polymer moiety, amino, substituted amino, alkyl and substituted        alkyl wherein each amino, substituted amino, alkyl and        substituted alkyl is optionally covalently bound to a polymer        moiety wherein, in each case, the polymer moiety optionally        comprises a linker which covalently links the polymer moiety;    -   Ar¹ is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl;    -   Ar² is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl wherein each of        aryl, substituted aryl, heteroaryl and substituted heteroaryl is        optionally covalently bound to a polymer moiety wherein the        polymer moiety optionally comprises a linker which covalently        links the polymer moiety to Ar²;    -   R² and R³ are independently selected from the group consisting        of hydrogen, alkyl, substituted alkyl, and where R² and R³,        together with the nitrogen atom bound thereto, form a        heterocyclic ring or a substituted heterocyclic ring wherein        each of alkyl, substituted alkyl, heterocyclic and substituted        heterocyclic is optionally covalently bound to a polymer moiety        which further optionally comprises a linker;    -   n is an integer equal to 0, 1 or 2; and    -   pharmaceutically acceptable salts thereof;    -   provided that at least one of R, Ar², and —NR²R³ is covalently        bound to a polymer which optionally comprises a linker;    -   and further provided that the conjugate of formula Id has a        molecular weight of no more than about 80,000.

Preferred conjugates of formula I include those of formula Ie below:

wherein each A is independently a compound of formula IIe below:

and wherein q is 2 to about 20;

-   -   B is as defined above;    -   Ar¹ is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl;    -   Ar² is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl wherein each of        aryl, substituted aryl, heteroaryl and substituted heteroaryl is        optionally covalently bound to a polymer moiety wherein the        polymer moiety optionally comprises a linker which covalently        links the polymer moiety to Ar²;    -   R² and R³ are independently selected from the group consisting        of hydrogen, alkyl, substituted alkyl, and where R² and R³,        together with the nitrogen atom bound thereto, form a        heterocyclic ring or a substituted heterocyclic ring wherein        each of alkyl, substituted alkyl, heterocyclic and substituted        heterocyclic is optionally covalently bound to a polymer moiety        which further optionally comprises a linker; and    -   pharmaceutically acceptable salts thereof;    -   provided that at least one of Ar² and —NR²R³ is covalently bound        to a polymer moiety which optionally comprises a linker;    -   and further provided that the conjugate of formula Ie has a        molecular weight of not more than 60,000.

Preferred conjugates of formula I include those of formula If below:

wherein each A is independently a compound of formula IIf below:

-   -   and wherein q is 2 to about 20;    -   B is as defined above;    -   R⁴ is covalently bound to a polymer moiety which optionally        comprises a linker;    -   R⁵ is selected from the group consisting of alkyl and        substituted alkyl;    -   Ar³ is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl;    -   X is selected from the group consisting of —NR¹—, —O—, —S—,        —SO—, —SO₂ and optionally substituted —CH₂— where R¹ is selected        from the group consisting of hydrogen and alkyl;    -   m is an integer equal to 0, 1 or 2;    -   n is an integer equal to 0, 1 or 2; and    -   pharmaceutically acceptable salts thereof;    -   provided that:    -   A. when R is covalently bound to the polymer moiety, n is one        and X is not —O—, —S—, —SO—, or —SO₂—;    -   B. when X is —O— or —NR¹—, then m is two; and    -   C. the conjugate of formula If has a molecular weight of no more        than 60,000.

Preferred conjugates of formula I include those of formula Ig below:

-   -   wherein each A is independently a compound of formula IIg below:    -   and wherein q is 2 to about 20;    -   B is as defined above;    -   R⁴ is covalently bound to a polymer moiety which optionally        comprises a linker;    -   R⁵ is selected from the group consisting of alkyl and        substituted alkyl;    -   Ar³ is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl;    -   n is an integer equal to 0, 1 or 2; and    -   pharmaceutically acceptable salts thereof;    -   provided that the conjugate of formula Ig has a molecular weight        of not more than 60,000.

Preferred conjugates of formula I include those of formula Ih below:

-   -   wherein each A is independently a compound of formula IIh below:    -   and wherein q is 2 to about 20;    -   R⁴ is covalently bound to a polymer moiety which optionally        comprises a linker;    -   Ar³ is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl;    -   pharmaceutically acceptable salts thereof;    -   provided that the conjugate of formula Ih has a molecular weight        of not more than 60,000.

Preferred conjugates of formula I include those of formula Ii below:

wherein each A is independently a compound of formula IIi below:

or a pharmaceutically acceptable salt thereof,

-   -   and wherein q is 2 to about 20;    -   and provided that the conjugate of formula Ii has a molecular        weight of no more than 60,000.

Preferred conjugates of formula I include those of formula Ij below:

wherein each A is independently a compound of formula lIj below:

or a pharmaceutically acceptable salt thereof,and wherein q is about 2 to about 20;

-   -   and provided that the conjugate of formula Ij has a molecular        weight of no more than about 80,000.

Preferred conjugates of formula I include those of formula Ik below:

wherein each A is independently a compound of formula IIk below:

-   -   or a pharmaceutically acceptable salt thereof.

Preferred conjugates of formula I include those of formula IL below:

-   -   wherein each A is independently a compound of formula IIL below:        or a pharmaceutically acceptable salt thereof, wherein

-   R⁴ is covalently bound to the polymer moiety which optionally    comprises a linker.

Preferably, Ar¹ in formulae IIa-IIe and Ar³ in formulae IIf-IIh areindependently selected from the group consisting of:

-   phenyl,-   4-methylphenyl,-   4-t-butylphenyl,-   2,4,6-trimethylphenyl,-   2-fluorophenyl,-   3-fluorophenyl,-   4-fluorophenyl,-   2,4-difluorophenyl,-   3,4-difluorophenyl,-   3,5-difluorophenyl,-   2-chlorophenyl,-   3-chlorophenyl,-   4-chlorophenyl,-   3,4-dichlorophenyl,-   3,5-dichlorophenyl,-   3-chloro-4-fluorophenyl,-   4-bromophenyl,-   2-methoxyphenyl,-   3-methoxyphenyl,-   4-methoxyphenyl,-   3,4-dimethoxyphenyl,-   4-t-butoxyphenyl,-   4-(3′-dimethylamino-n-propoxy)-phenyl,-   2-carboxyphenyl,-   2-(methoxycarbonyl)phenyl,-   4-(H₂NC(O)-)phenyl,-   4-(H₂NC(S)-)phenyl,-   4-cyanophenyl,-   4-trifluoromethylphenyl,-   4-trifluoromethoxyphenyl,-   3,5-di-(trifluoromethyl)phenyl,-   4-nitrophenyl,-   4-aminophenyl,-   4-(CH₃C(O)NH-)phenyl,-   4-(phenylNHC(O)NH-)phenyl,-   4-amidinophenyl,-   4-methylamidinophenyl,-   4-[CH₃SC(═NH)-]phenyl,-   4-chloro-3-[H₂NS(O)₂-]phenyl,-   1-naphthyl,-   2-naphthyl,-   pyridin-2-yl,-   pyridin-3-yl,-   pyridin4-yl,-   pyrimidin-2-yl,-   quinolin-8-yl,-   2-(trifluoroacetyl)-1,2,3,4-tetrahydroisoquinolin-7-yl,-   2-thienyl,-   5-chloro-2-thienyl,-   2,5-dichloro-4-thienyl,-   1-N-methylimidazol-4-yl,-   1-N-methylpyrazol-3-yl,-   1-N-methylpyrazol-4-yl,-   1-N-butylpyrazol-4-yl,-   1-N-methyl-3-methyl-5-chloropyrazol-4-yl,-   1-N-methyl-5-methyl-3-chloropyrazol-4-yl,-   2-thiazolyl and-   5-methyl-1,3,4-thiadiazol-2-yl.

Preferably, when A is of the formulae IIa, IIb, IIc, IId, and IIe, andAr¹ is bound to a polymer moiety, then Ar¹ is of the formula:—Ar¹—Z—(CH₂CHR⁷O)_(p)R⁸

-   -   wherein    -   Ar¹ is selected from the group consisting of aryl, substituted        aryl, heteroaryl, and substituted heteroaryl,    -   Z is selected from the group consisting of a covalent bond, a        linking group of from 1 to 40 atoms, —O—, and —NR⁹—, where R⁹ is        selected from the group consisting of hydrogen and alkyl,    -   R⁷ is selected from the group consisting of hydrogen and methyl;    -   R⁸ is selected from the group consisting of -(L)_(W)-A when p is        greater than about 300 and (L)-B-(A)_(q-1), wherein A is        represented by any of formulae ILa through IIh above, L is a        linking group of from 1 to 40 atoms and w is zero or one: and    -   p is an integer of from about 200 to 1360.

When A is of the Formulae IIa or IIf, and R is not bound to a polymermoiety, the substituent of the following formula:

-   -   where R⁵, X, m and n are as defined above, is preferably        selected from the group consisting of azetidinyl, thiazolidinyl,        piperidinyl, piperazinyl, morpholino, thiomorpholinyl,        pyrrolidinyl, 4-hydroxypyrrolidinyl, 4-oxopyrrolidinyl,        4-fluoropyrrolidinyl, 4,4-difluoropyrrolidinyl,        4-(thiomorpholin-4-ylC(O)O-)pyrrolidinyl,        4-[CH₃S(O)₂O-]pyrrolidinyl, 3-phenylpyrrolidinyl,        3-thiophenylpyrrolidinyl, 4-amino-pyrrolidinyl,        3-methoxypyrrolidinyl, 4,4-dimethylpyrrolidinyl,        4-N-Cbz-piperazinyl, 4-[CH₃S(O)₂-]piperazinyl,        5,5-dimethylthiazolindin-4-yl, 1,1-dioxo-thiazolidinyl,        1,1-dioxo-5,5-dimethylthiazolidin-2-yl and        1,1-dioxothiomorpholinyl.

Preferably, when A is of the formulae IIa and the substituent of theformula:

-   -   is bound to the polymer moiety, then preferably the substituent        is of the formula:    -   wherein    -   m is an integer equal to zero, one or two;    -   Z is selected from the group consisting of a covalent bond, a        linking group of from 1 to 40 atoms, —O—, —NR⁹—, where R⁹ is        selected from the group consisting of hydrogen and alkyl,    -   R⁷ is selected from the group consisting of hydrogen and methyl;    -   p is an integer of from 0 to about 1360;    -   R⁸ is selected from the group consisting of -B-(A)_(q-1), and A        when p is greater than about 300, and A is represented by any of        formulae IIa through IIh above.

When A is of the formula IIa, IIb, IIc, IId, IIe and when Ar² is notbound to a polymer moiety, then preferably Ar² is selected from thegroup consisting of phenyl, substituted phenyl, 2-pyridinyl,3-pyridinyl, 4-pyridinyl, and 4-pyridin-2-onyl.

When A is of the formula IIa, IIb, IIc, IId, IIe and when Ar² is boundto a polymer moiety, then Ar² is preferably represented by the formula:

-   -   where Ar² is selected from the group consisting of aryl,        substituted aryl, heteroaryl and substituted heteroaryl;    -   Z is selected from the group consisting of a covalent bond, a        linking group of from 1 to 40 atoms, —O—, —NR⁹—, amide,        carbamate and urea, where R⁹ is selected from the group        consisting of hydrogen and alkyl,    -   R⁷ is selected from the group consisting of hydrogen and methyl;    -   p is an integer of from 0 to about 1360;    -   R⁸ is selected from the group consisting of -B-(A)_(q-1), and A        when p is greater than about 300, and A is represented by any of        formulae IIa through IIh above.

In one preferred embodiment, —YC(O)W is —OC(O)NR²R³.

When A is of the formulae IIa, IIb, or IIc, —YC(O)W is —OC(O)NR²R³ andneither R² nor R³ are bound to a polymer moiety, then preferably—OC(O)NR²R³ is selected from the group consisting of:

-   (CH₃)₂NC(O)O—,-   (piperidin-1-yl)-C(O)O—,-   (piperidin-4-yl)-C(O)O—,-   (1-methylpiperidin-4-yl)-C(O)O—,-   (4-hydroxypiperidin-1-yl)-C(O)O—,-   (4-formyloxypiperidin-1-yl)-C(O)O—,-   (4-ethoxycarbonylpiperidin-1-yl)-C(O)O—,-   (4-carboxylpiperidin-1-yl)-C(O)O—,-   (3-hydroxymethylpiperidin-1-yl)-C(O)O—,-   (4-hydroxymethylpiperidin-1-yl)-C(O)O—,-   (4-phenyl-1-Boc-piperidin-4-yl)-C(O)O—,-   (4-piperidon-1-yl ethylene ketal)-C(O)O—,-   (piperazin-4-yl)-C(O)O—,-   (1-Boc-piperazin-4-yl)-C(O)O—,-   (4-methylpiperazin-1-yl)-C(O)O—,-   (4-methylhomopiperazin-1-yl)-C(O)O—,-   (4-(2-hydroxyethyl)piperazin-1-yl)-C(O)O—,-   (4-phenylpiperazin-1-yl)-C(O)O—,-   (4-(pyridin-2-yl)piperazin-1]-yl)-C(O)O—,-   (4-(4-trifluoromethylpyridin-2-yl)piperazin-1-yl)-C(O)O—,-   (4-(pyrimidin-2-yl)piperazin-1-yl)-C(O)O—,-   (4-acetylpiperazin-1-yl)-C(O)O—,-   (4-(phenyl-C(O)-)piperazin-1-yl)-C(O)O—,-   (4-(pyridin4′-yl-C(O)-)piperazin-1-yl)-C(O)O—,-   (4-(phenyl-NHC(O)-)piperazin-1-yl)-C(O)O—,-   (4-(phenyl-NHC(S)-)piperazin-1-yl)-C(O)O—,-   (4-methanesulfonylpiperazin-1-yl)-C(O)O—,-   (4-trifluoromethanesulfonylpiperazin-1-yl)-C(O)O—,-   (morpholin-4-yl)-C(O)O—,-   (thiomorpholin-4-yl)-C(O)O—,-   (thiomorpholin-4′-yl sulfone)-C(O)O—,-   (pyrrolidin-1-yl)-C(O)O—,-   (2-methylpyrrolidin-1-yl)-C(O)O—,-   (2-(methoxycarbonyl)pyrrolidin-1-yl)-C(O)O—,-   (2-(hydroxymethyl)pyrrolidin-1-yl)-C(O)O—,-   (2-(N,N-dimethylamino)ethyl)(CH₃)NC(O)O—,-   (2-(N-methyl-N-toluene-4-sulfonylamino)ethyl)(CH₃)N—C(O)O—,-   (2-(morpholin-4-yl)ethyl)(CH₃)NC(O)O—,-   (2-(hydroxy)ethyl)(CH₃)NC(O)O—,-   bis(2-(hydroxy)ethyl)NC(O)O—,-   (2-(formyloxy)ethyl)(CH₃)NC(O)O—,-   (CH₃OC(O)CH₂)HNC(O)O—, and-   2-(phenylNHC(O)O—)ethyl-]HNC(O)O—.

When A is of the formulae IIa, IIb, or IIc, —YC(O)W is —OC(O)NR²R³ andR² and/or R³ are/is bound to the polymer moiety, the polymer moiety ispreferably represented by the formula:—Z¹—(CH₂CHR⁷O)_(p)R⁸

-   -   Z is selected from the group consisting of a covalent bond, a        linking group of from 1 to 40 atoms, —O—, —NR⁹—, amide,        carbamate and urea, where R⁹ is selected from the group        consisting of hydrogen and alkyl,    -   R⁷ is selected from the group consisting of hydrogen and methyl;    -   p is an integer of from 0 to about 1360;    -   R⁸ is selected from the group consisting of -B-(A)_(q-1), and A        when p is greater than about 300, and A is represented by any of        formulae IIa through IIh above.

In the compounds of formula IIi and IIj, it is preferred that that thegroup of

is of the formula:

wherein R⁵⁶ is a covalent bond to the polymer moiety which optionallycomprises a linker, or R⁶⁶ is hydrogen or straight or branchedC₁-C₆alkyl; R⁷⁷ is a covalent bond to a polymer moiety which optionallycomprises a linker, or R⁷⁷ is hydrogen, halogen or straight or branchedC₁-C₆alkoxy; and R⁸⁸ is a covalent bond to the polymer moiety whichoptionally comprises a linker, or R⁸⁸ is hydrogen or straight orbranched C_(1-C) ₆alkyl Preferably, one of R⁶⁶, R⁷⁷, and R⁸⁸ is acovalent bond to the polymer moiety which optionally comprises a linker.

Preferred compounds of formula IIi are also those of the formula IIi-a:

and pharmaceutically acceptable salts thereof, wherein

-   Ar¹ is selected from the group consisting of alkyl, substituted    alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,    heterocyclic, substituted heterocyclic, heteroaryl and substituted    heteroaryl; and-   R⁶ is a covalent bond to a polymer moiety which optionally comprises    a linker.

Preferred compounds of Formula IIi-a include those wherein Ar¹ is phenylor a 5- or 6-membered heteroaryl group having at least one nitrogenatom, each of which is optionally substituted with halogen, hydroxy,C₁-C₆ alkoxy, C₁-C₆ alkyl, nitro, trifluoromethyl, amino, mono- ordi(C₁-C₆)alkylamino, amino(C₁-C₆)alkyl, C₂-C₆ acyl, C₂-C₆ acylamino, oramino(C₁-C₆)acyl. Ar¹ is pyridyl optionally substituted with halogen,hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy, nitro, trifluoromethyl, amino, mono-or di(C₁-C₆)alkylamino, amino(C₁-C₆)alkyl, C₂-C₆ acyl, C₂-C₆ acylamino,or amino(C₁-C₆)acyl. Particularly preferred compounds of Formula IIi-ainclude those where Ar¹ is pyridyl optionally substituted with C₁-C₆alkyl, hydroxy, halogen, C₁-C₆ alkoxy, nitro, trifluoromethyl, amino, ormono- or di(C₁-C₆)alkylamino.

Preferred compounds of formula IIj are also those of the formula IIj-a:

and pharmaceutically acceptable salts thereof, wherein

-   R⁶ is a covalent bond to a polymer moiety which optionally comprises    a linker.

Preferred compounds of Formula IIj-a include those where R³¹ is amino ormono- or di(C₁-C₆)alkylamino; and R³² is —H, —NO₂ or haloalkyl, morepreferably trifluoromethylmethyl.

Still other preferred compounds of Formula IIj-a are those where

-   R³¹ is amino or mono- or di(C₁-C₆)alkylamino; and-   R³² is —N(MR⁴¹)R⁴²; where M is —SO₂— or —CO—;-   R⁴¹ is C₁-C₆ alkyl optionally substituted with halogen, hydroxy,    C₁-C₆ alkoxy, amino, or mono- or di(C₁-C₆)alkylamino; or    -   phenyl or a 5- or 6-membered heteroaryl containing at least one        nitrogen, each of which is optionally substituted with halogen,        hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₇ cycloalkyl, amino,        nitro, trifluoromethyl, or    -   mono- or di(C₁-C₆)alkylamino; and-   R⁴² is hydrogen, C₁-C₆alkyl, or C₃-C₇cycloalkyl.

Further preferred compounds of formula II-j-a include those wherein

-   R⁴¹ groups within Formula IIj-a are C₁-C₄ alkyl optionally    substituted with halogen, hydroxy, C₁-C₆ alkoxy, amino, or mono- or    di(C₁-C₆)alkylamino; or    -   pyridyl or pyrimidinyl, each of which is optionally substituted        with halogen, hydroxy, C₁-C₃ alkyl, C₁-C₃ alkoxy, amino, or        mono- or di(C₁-C₄)alkylamino; and-   R⁴² is hydrogen, C₁-C₄alkyl, or C₃-C₇cycloalkyl.

In one example, the conjugates of this invention are divalent and arerepresented by formula III:

-   -   where each A is independently as defined above and B′ is        —Z′—(CH₂CHR⁷O)_(p)—Z′— where each Z′ is independently a covalent        bond or a linking group, R⁷ is hydrogen or methyl and p is an        integer of from about 100 to 1360.

In another example, the conjugates of this invention are trivalent todecavalent and are preferably represented by formula IV:

-   -   where each A is independently as defined above and t is an        integer from 3 to 10.        Definitions

As used herein, “alkyl” refers to monovalent saturated aliphatichydrocarbyl groups having from 1 to 5 carbon atoms and more preferably 1to 3 carbon atoms. This term is exemplified by groups such as methyl,ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, n-pentyl and the like.

“Substituted alkyl” refers to an alkyl group having from 1 to 3, andpreferably 1 to 2, substituents selected from the group consisting ofalkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy,cyano, halogen, hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl,substituted cycloalkyl, spirocycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic.

“Optionally substituted alkyl” encompasses “alkyl” and “substitutedalkyl” as defined above.

“Optionally substituted —CH₂—” refers to a group that is eitherunsubstituted or is substituted with 1 or 2 substituents selected fromthe group consisting of alkoxy, substituted alkoxy, thioalkoxy,substituted thioalkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy,cyano, halogen, hydroxyl, sulfhydryl, nitro, carboxyl, carboxyl esters,cycloalkyl, substituted cycloalkyl, spirocycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic.Preferred substituents on “optionally substituted —CH₂—” are hydroxy,alkoxy, amino, monoalkyl amino, dialkylamino, sulfhydryl, thioalkoxy,and halogen (preferably F). Preferably, when “optionally substituted—CH₂—” is substituted, it is substituted with one group.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupspreferably having from 1 to 5 and more preferably 1 to 3 carbon atomswhich are either straight-chained or branched. This term is exemplifiedby groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—) and the like.

“Alkoxy” refers to the group “alkyl-O-” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy,sec-butoxy, n-pentoxy and the like.

“Substituted alkoxy” refers to the group “substituted alkyl-O—”.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)-cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Aminoacyl” refers to the group —C(O)NR¹⁰R¹⁰ where each R¹⁰ isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic and where each R¹⁰ is joined to form together with thenitrogen atom a heterocyclic or substituted heterocyclic ring whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, substitutedheteroaryl-C(O)O—, heterocyclic-C(O)O—, and substitutedheterocyclic-C(O)O— wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Alkenyl” refers to alkenyl groups having from 2 to 6 carbon atoms andpreferably 2 to 4 carbon atoms and having at least 1 and preferably from1 to 2 sites of alkenyl unsaturation. Such groups are exemplified byvinyl, allyl, but-3-en-1-yl, and the like.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic withthe proviso that any hydroxyl substitution is not attached to a vinyl(unsaturated) carbon atom.

“Alkynyl” refers to alkynyl groups having from 2 to 6 carbon atoms andpreferably 2 to 3 carbon atoms and having at least 1 and preferably from1 to 2 sites of alkynyl unsaturation.

“Substituted alkynyl” refers to alkynyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic.

“Amino” refers to the group —NH₂.

“Cyano” refers to the group —CN.

“Substituted amino” refers to the group —NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic and where R′ and R″ are joined, together with the nitrogenbound thereto to form a heterocyclic or substituted heterocyclic groupprovided that R′ and R″ are both not hydrogen. When R′ is hydrogen andR″ is alkyl, the substituted amino group is sometimes referred to hereinas alkylamino. When R′ and R″ are alkyl, the substituted amino group issometimes referred to herein as dialkylamino. When referring to amonosubstituted amino, it is meant that either R′ or R″ is hydrogen butnot both. When referring to a disubstituted amino, it is meant thatneither R′ or R″ is hydrogen.

“Nitro” refers to the group —NO₂.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryls includephenyl and naphthyl.

“Substituted aryl” refers to aryl groups which are substituted with from1 to 3 substituents, and preferably 1 to 2 substituents, selected fromthe group consisting of hydroxy, acyl, acylamino, acyloxy, alkyl,substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, amino, substituted amino,aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy,carboxyl, carboxyl esters, cyano, thiol, thioalkyl, substitutedthioalkyl, thioaryl, substituted thioaryl, thioheteroaryl, substitutedthioheteroaryl, thiocycloalkyl, substituted thiocycloalkyl,thioheterocyclic, substituted thioheterocyclic, cycloalkyl, substitutedcycloalkyl, halo, nitro, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, heteroaryloxy, substitutedheteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy, aminosulfonyl (NH₂—SO₂—), and substituted amino sulfonyl.

“Aryloxy” refers to the group aryl-O— that includes, by way of example,phenoxy, naphthoxy, and the like.

“Substituted aryloxy” refers to substituted aryl-O— groups.

“Carboxyl” refers to —COOH or salts thereof.

“Carboxyl ester” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)-aryl, and —C(O)O-substituted aryl wherein alkyl,substituted alkyl, aryl and substituted aryl are as defined herein.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including, by way of example,adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and thelike.

“Cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 10 carbonatoms having single or multiple cyclic rings and further having at least1 and preferably from 1 to 2 internal sites of ethylenic or vinyl(>C═C<) unsaturation.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refers to ancycloalkyl or cycloalkenyl group, having from 1 to 5 substituentsselected from the group consisting of oxo (═O), thioxo (═S), alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano,halogen, hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl,substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic, and substituted heterocyclic.

“Cycloalkoxy” refers to —O-cycloalkyl groups.

“Substituted cycloalkoxy” refers to —O-substituted cycloalkyl groups.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo andpreferably is fluoro or chloro.

“Hydroxy” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group.Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl, thiophenyl,and furanyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 3 substituents selected from the same groupof substituents defined for substituted aryl.

“Heteroaryloxy” refers to the group —O-heteroaryl and “substitutedheteroaryloxy” refers to the group —O-substituted heteroaryl.

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or unsaturated group having a single ring ormultiple condensed rings, from 1 to 10 carbon atoms and from 1 to 4hetero atoms selected from the group consisting of nitrogen, sulfur oroxygen within the ring wherein, in fused ring systems, one or more therings can be cycloalkyl, aryl or heteroaryl provided that the point ofattachment is through the heterocyclic ring.

“Substituted heterocyclic” or “substituted heterocycloalkyl” or“substituted heterocyclyl” refers to heterocyclyl groups that aresubstituted with from 1 to 3 of the same substituents as defined forsubstituted cycloalkyl.

Examples of heterocyclyls and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydro-isoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), piperidinyl, pyrrolidine, tetrahydrofuranyl, and thelike.

“Thiol” refers to the group —SH.

“Thioalkyl” or “alkylthioether” or “thioalkoxy” refers to the group—S-alkyl.

“Substituted thioalkyl” or “substituted alkylthioether” or “substitutedthioalkoxy” refers to the group —S-substituted alkyl.

“Thioaryl” refers to the group —S-aryl, where aryl is defined above.

“Substituted thioaryl” refers to the group —S-substituted aryl, wheresubstituted aryl is defined above.

“Thioheteroaryl” refers to the group —S-heteroaryl, where heteroaryl isas defined above.

“Substituted thioheteroaryl” refers to the group —S-substitutedheteroaryl, where substituted thioheteroaryl is defined above.

“Thioheterocyclic” refers to the group —S-heterocyclic and “substitutedthioheterocyclic” refers to the group —S-substituted heterocyclic, whereheterocyclic and substituted heterocyclic.

“Heterocyclyloxy” refers to the group heterocyclyl-O— and “substitutedheterocyclyl-O— refers to the group substituted heterocyclyl-O— whereheterocyclyl and substituted heterocyclyl are as defined above.

“Thiocycloalkyl” refers to the group —S-cycloalkyl and “substitutedthiocycloalkyl” refers to the group —S-substituted cycloalkyl, wherecycloalkyl and substituted cycloalkyl are as defined above.

“Hydrolyzable ester” refers to a group that is hydrolyzed in vivo toproduce the the parent acid. Examples of such groups include alkoxy,substituted alkoxy, cycloalkoxy, substituted cycloalkoxy, aryloxy andsubstituted aryloxy. A preferred hydrolyzable ester is alkoxy.

“Hydrolyzable polymer ester” refers to a biocompatible polymer that ishydrolyzed in vivo to produce the the parent acid. A preferredhydrolyzable polymer ester is PEG.

The terms “compound” and “active compound” are used to refer to theVLA-4 antagonist portion of a conjugate of the invention or to a VLA-4antagonist as it exists prior to conjugation to a polymer.

The terms “Linker”, “linking group” or “linker of from 1 to 40 atoms”refer to a group or groups that (1) covalently links the polymer to theactive compound and/or (2) covalently link the polymer, oligomer, and/ormonomer portions comprising the polymer moiety one to another;non-limiting illustrations of the latter include polymer-linker-polymer,oligomer-linker-oligomer, monomer-linker-monomer,oligomer-linker-oligomer-linker, and the like. Within any particularconjugate, the linker connecting the polymer, oligomer, and/or monomerportions of a polymer moiety together, and the linker bonding a polymermoiety to an active compound may be the same or different (i.e., mayhave the same or different chemical structures).

The linker that covalently links the polymer, oligomer, and/or monomerportions, such as polyalkylene oxide portions, of a polymer moiety oneto another is also referred to as a “branched-arm hub”, or “branched-armhub molecule”. Branched-arm hubs are molecules that covalently bondthree or more polymer, oligomer, and/or monomer portions to them,providing tri-valent or higher valent polymer moieties for conjugationwith the active compound. Non-limiting examples of such hub moleculesare glycerol (1,2,3-propanetriol), pentaerythitol, lysine,1,2,4-benzenetriol, glucose (in its pyranose form), ethylenediaminetetraacetic acid, amino acids, 3- or 4-aminosalicylic acid,1,3-diamino-2-hydroxypropane, glucosamine, and sialic acid.

Representative functional group linkages, of which a linking group mayhave one or more, are amides (—C(O)NR³—), ethers (—O—), thioethers(—S—), carbamates (—OC(O)NR³—), thiocarbamates (—OC(S)NR³—), ureas(—NR³C(O)NR³—), thioureas (—NR³C(S)NR³—), amino groups (—NR³—), carbonylgroups (—C(O)—), alkoxy groups (—O-alkylene-), etc. The linker may behomogenous or heterogeneous in its atom content (e.g., linkerscontaining only carbon atoms or linkers containing carbon atoms as wellas one or more heteroatoms present on the linker. Preferably, the linkercontains 1 to 25 carbon atoms and 0 to 15 heteroatoms selected fromoxygen, NR³, sulfur, —S(O)— and —S(O)₂—, where R³ is hydrogen, alkyl orsubstituted alkyl. The linker may also be chiral or achiral, linear,branched or cyclic.

Intervening between the functional group linkages or bonds within thelinker, the linker may further contain spacer groups including, but notlimited to, spacers selected from alkyl, substituted alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, substituted heterocyclic, andcombinations thereof. The spacer may be homogenous or heterogeneous inits atom content (e.g., spacers containing only carbon atoms or spacerscontaining carbon atoms as well as one or more heteroatoms present onthe spacer. Preferably, the spacer contains 1 to 25 carbon atoms and 0to 15 heteroatoms selected from oxygen, NR³, sulfur, —S(O)— and —S(O)₂—,where R³ is as defined above. The spacer may also be chiral or achiral,linear, branched or cyclic.

Non-limiting examples of spacers are straight or branched alkylenechains, phenylene, biphenylene, etc. rings, all of which are capable ofcarrying one or more than one functional group capable of forming alinkage with the active compound and one or more polyalkylene oxidemoieties. One particular example of a polyfunctional linker-spacer groupis lysine, which may link any of the active compounds to two polymermoieties via the two amino groups substituted on a C₄ alkylene chain.Other non-limiting examples include p-aminobenzoic acid and3,5-diaminobenzoic acid which have 2 and 3 functional groupsrespectively available for linkage formation. Other such polyfunctionallinkage plus spacer groups can be readily envisaged by one of skill inthe art.

The terms “polymer” and “polymer moiety” refers to biocompatible,water-soluble, substantially non-immunogenic, polymers which are capableof being coupled to more than one VLA-4 antagonist of formula II.Preferably the polymer is non-ionic and biocompatible as measured bylack of toxicity at the molecular weights and dosages used. The termsalso encompass molecules in which 3 or more polymers are connected to abranched-arm hub molecule, as discussed above. The terms furtherencompass molecules in which the polymer, oligomer, and/or monomerportions thereof are connected by one or more linkers.

Examples of suitable polymers include, but are not limited to:polyoxyalkylene polymers such as polyethylene glycol (PEG),polyvinylpyrrolidone (PVP), polyacrylamide (PAAm),polydimethylacrylamide (PDAAm), polyvinyl alcohol (PVA), dextran, poly(L-glutamic acid) (PGA), styrene maleic anhydride (SMA),poly-N-(2-hydroxypropyl) methacrylamide (HPMA), polydivinylether maleicanhydride (DIVEMA) (Kameda, Y. et al., Biomaterials 25: 3259-3266, 2004;Thanou, M. et al, Current Opinion in Investigational Drugs 4(6):701-709, 2003; Veronese, F. M., et al., II Farmaco 54: 497-516, 1999).

Preferred polymers are polyoxyalkylenes. By “polyoxyalkylenes” is meantmacromolecules that include at least one polyalkylene oxide portion thatis optionally covalently bonded to one or more additional polyakyleneoxides, wherein the polyalkylene oxides are the same or different.Non-limiting examples include polyethylene glycol (PEG), polypropyleneglycol (PPG), polyisopropylene glycol (PIPG), PEG-PEG, PEG-PPG,PPG-PIPG, and the like. Also included within the definition ofpolyoxyalkylenes are macromolecules wherein the polyalkylene oxideportions are optionally connected to each other by a linker.Illustrative examples are PEG-linker-PEG, PEG-linker-PIPG, and the like.More specific examples include the commercially availablepoly[di(ethylene glycol)adipates, poly[di(ethylene glycol)phthalatediols, and the like. Other examples are block copolymers of oxyalkylene,polyethylene glycol, polypropylene glycol, and polyoxyethylenated polyolunits.

At least one of its termini, the polymer is covalently attached tonon-polymer substituted compound of Formula II optionally through alinker using conventional chemical techniques providing for covalentlinkage of the polymer to the non-polymer substituted compound ofFormula II.

When a linker is employed, the linker is covalently bonded to at leastone of the polymer termini which, in turn, is covalently attached to theotherwise, non-polymer substituted compound of Formula II. Reactionchemistries resulting in such linkages are well known in the art. Suchreaction chemistries involve the use of complementary functional groupson the linker, the non-polymer substituted compound of Formula II andthe polymer. Preferably, the complementary functional groups on thelinker are selected relative to the functional groups available on thepolymer for bonding or which can be introduced onto the polymer forbonding. Again, such complementary functional groups are well known inthe art. For example, reaction between a carboxylic acid of either thelinker or the polymer and a primary or secondary amine of the polymer orthe linker in the presence of suitable, well-known activating agentsresults in formation of an amide bond covalently linking the polymermoiety to the linker; reaction between an amine group of either thelinker or the polymer group and a sulfonyl halide of the polymer or thelinker results in formation of a sulfonamide bond covalently linking thepolymer moiety to the linker; and reaction between an alcohol or phenolgroup of either the linker or the polymer and an alkyl or aryl halide ofthe polymer or the linker results in formation of an ether bondcovalently linking the polymer group to the linker.

It is understood, of course, that if the appropriate substituents arefound on the non-polymer substituted compound of Formula II then theoptional linker may not be needed as there can be direct linkage of thepolymer to the non-polymer substituted compound of Formula II.

Table I below illustrates numerous complementary reactive groups and theresulting bonds formed by reaction there between. One of ordinary skillin the art can select the appropriate solvents and reaction conditionsto effect these linkages. TABLE I Representative Complementary BindingChemistries First Reactive Group Second Reactive Group Linkage HydroxylIsocyanate Urethane Amine Epoxide β-hydroxyamine sulfonyl halide AmineSulfonamide Carboxyl Amine Amide Hydroxyl alkyl/aryl halide EtherAldehyde Amine Amine (under reductive amination conditions)

Preferred linkers include, by way of example, the following —O—, —NR³—,—NR³C(O)O—, —OC(O)NR³—, —NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—,-alkylene-NR³C(O)O—, -alkylene-NR³C(O)NR³—, -alkylene-OC(O)N R³—,-alkylene-NR³—, -alkylene-O—, -alkylene-NR³C(O)—, -alkylene-C(O)NR³—,—NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)N R³-alkylene,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-, —C(O)NR³-alkylene-,-alkylene-NR³C(O)O-alkylene-, -alkylene-NR³C(O)NR³-alkylene-,-alkylene-OC(O)NR³-alkylene-, -alkylene-NR³-alkylene-,alkylene-O-alkylene-, -alkylene-NR³C(O)-alkylene-, —C(O)NR³-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy, —NR³-alkyleneoxy-, —O-alkyleneoxy-,—NR³C(O)-alkyleneoxy-, —C(O)NR³-alkyleneoxy-,-alkyleneoxy-NR³C(O)O-alkyleneoxy- where R³ is as defined above and

where

is selected from the group consisting of aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic, and D and E are independentlyselected from the group consisting of a bond, —O—, CO, —NR³—,—NR³C(O)O—, —OC(O)NR³—, —NR³C(O)—, —C(O)NR³—, —NR³C(O)NR³—,-alkylene-NR³C(O)O—, -alkylene-NR³C(O)NR³—, -alkylene-OC(O)NR³—,-alkylene-NR³—, -alkylene-O—, -alkylene-NR³C(O)—, alkylene-C(O)NR³—,—NR³C(O)O-alkylene-, —NR³C(O)NR³-alkylene-, —OC(O)NR³-alkylene-,—NR³-alkylene-, —O-alkylene-, —NR³C(O)-alkylene-,—NR³C(O)O-alkyleneoxy-, —NR³C(O)NR³-alkyleneoxy-, —OC(O)NR³-alkyleneoxy,—NR³-alkyleneoxy-, —O-alkyleneoxy-, —NR³C(O)-alkyleneoxy-,—C(O)NR³-alkyleneoxy-, -alkyleneoxy-NR³C(O)O-alkyleneoxy-,—C(O)NR³-alkylene-, -alkylene-NR³C(O)O-alkylene-, -alkylene-NR³C(O)NR³-alkylene-, -alkylene-OC(O) NR³-alkylene-,-alkylene-NR³-alkylene-, alkylene-O-alkylene-, -alkylene-NR³C(O)-alkylene-, and —C(O)NR³-alkylene-, where R³ is as defined above.

Preferred alkylene groups in the above linkers include C₁-C₁₅ alkylenegroups, more preferably C₁-C₆ alkylene groups, and most preferably C₁-C₃alkylene groups. Preferred heterocyclic groups include piperazinyl,piperidinyl, homopiperazinyl, homopiperidinyl, pyrrolidinyl, andimidazolidinyl. Preferred alkoxy groups are —(CH₂—CH₂—O)₁₋₁₅.

The term “oxyalkylene” refers to —OCH₂CHR^(d) where R^(d) is alkyl.Polymerized oxyalkylenes are referred to as polyoxyalkylenes,polyalkylene oxides or polyalkylene glycols, non-limiting examples ofwhich include PEG, poly propylene glycol, polybutylene glycol,polyisopropylene glycol, and the like.

Such polymers are optionally mono-capped with a substituent preferablyselected from alkyl, aryl, substituted alkyl, substituted aryl and a abranched-arm hub molecule as described above. Inclusive of such polymersare those diamino capped polyoxyalkylene polymers which are known in theart as Jeffamines®. Generally, Jeffamines® (available from HuntsmanPerformance Products, The Woodlands, Tex.) contain primary amino groupsattached to the terminus of a polyether backbone. They are thus“polyether amines.” The polyether backbone is based either on propyleneoxide (PO), ethylene oxide (EO), or mixed EO/PO, or other backbonesegments. Jeffamines® may be monoamines, diamines, and triamines, andare available in a variety of molecular weights, ranging up to about5,000.

Still further, polymerized oxyalkylenes can optionally contain one ormore non-oxyalkylene units such as the commercially availablepoly[di(ethylene glycol)adipates, poly[di(ethylene glycol)phthalatediols, and the like. Also included are block copolymers of oxyalkylene,polyethylene glycol, polypropylene glycol, and polyoxyethylenated polyolunits.

Polyoxyalkylenes, such as PEG, are usually provided as a water soluble,waxy solid. Generally, as the polymer's molecular weight increases, itsviscosity and freezing point also increase. Commercial preparations areusually characterized by the “average molecular weight” of theconstituent polymers.

Typically, the average molecular weight of the total amount of polymerarising from single or multiple polymer moieties in the conjugates ofthe invention is between about 100 to 100,000; preferably from about20,000 to 60,000; more preferably from about 30,000 to about 50,000. Itis apparent to those skilled in the art that polymers of this type willbe polydisperse. Polydispersity refers to the fact that polymermolecules, even ones of the same type, come in different sizes (chainlengths, for linear or multi-armed polymers). Therefore averagemolecular weight will depend on the method of averaging. Thepolydispersity index, a common measure of the variability of molecularweights is the ratio of the weight average molecular weight to thenumber average molecular weight. It indicates the distribution ofindividual molecular weights in a batch of polymers. The number averagemolecular weight is a way of determining the molecular weight of apolymer. The number average molecular weight is the common average ofthe molecular weights of the individual polymers. It is determined bymeasuring the molecular weight of n polymer molecules, summing theweights, and dividing by n. The number average molecular weight of apolymer can be determined by osmometry, end-group titration, andcolligative properties.

The weight average molecular weight can be determined by lightscattering, small angle neutron scattering (SANS), X-ray scattering, andsedimentation velocity. The ratio of the weight average to the numberaverage is called the polydispersity index. A theoretical sample ofpolymer having no dispersity would have a polydispersity index of 1.Preferred range of polydispersity index for the present invention isfrom about 1.10 to about 1.05. More preferred is a range from about 1.05to the upper limit of commercially feasible synthesis, which to date isabout 1.02.

Other suitable polymers such as polyvinylpyrrolidone (PVP),polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), polyvinyl alcohol(PVA), dextran, poly (L-glutamic acid) (PGA), styrene maleic anhydride(SMA), poly-N-(2-hydroxypropyl) methacrylamide (HPMA), polydivinylethermaleic anhydride (DIVEMA) are well known in the art and have molecularweights of from about 100 to 100,000; preferably from about 10,000 to80,000; more preferably from about 20,000 to about 70,000.

Non-limiting examples of PEGs that can be used in the invention includethe following: HO(alkylene-O)_(pp)R^(bb) mono-capped mono-hydroxy PEG(mPEG) H₂N(alkylene-O)_(pp)R^(bb) mono-capped mono-amino PEGHO(alkylene-O)_(pp)R—OH non-capped di-hydroxy PEGH₂N(alkylene-O)_(pp)R—OH non-capped mono-amino PEGHO(alkylene-O)_(pp)R^(bb) branched mono-hydroxy PEGHO(alkylene-O)_(pp)R^(bb) dendrimeric mono-hydroxy PEGwhere pp and alkylene are as defined herein and R is preferably selectedfrom the group consisting of alkyl, substituted alkyl, aryl andsubstituted aryl.Various PEG examples are shown below:

mono-capped mono-hydroxy PEG (mPEG)

mono-capped mono-amine PEG

non-capped di-hydroxy PEG

Branched PEGs:

-   -   PEG Reagents available from NOF (20 kDa 4-arm)

20 kDa 4-arm PEG tetra-amine

-   -   Diglycerine core    -   Cat # Sunbright DG-200PA    -   PEG Reagents available from Nektar (40 kDa 8-arm)

40 kDa 8-arm PEG

-   -   Hexaglycerine core    -   Cat # 0J000T08

Dendrimeric PEGs:

PEG Reagents available from NOF (40 kDa 4-arm)

40 kDa 4-arm PEG alcohol 40 kDa 4-arm PEG tetra-amine pentaerythritolcore pentaerythritol core cat. # Sunbright PTE-40000 cat. # SunbrightPTE-400PA

PEG Reagents available from NOF (40 kDa 3-arm)

40 kDa 3-arm PEG 40 kDa 3-arm PEG tri-amine glycerin core glycerin corecat. # Sunbright GL-40000 cat. #Sunbnght GL-400PA

-   -   PEG Reagents available from SunBio (40 and 20 kDa)

Y-PEG Series (Aspartic Acid Core)

6-Arm Series (Sorbitol Core)

Lower molecular weights available in the sorbitol 6-arm series include10, 15 and 20 kDa. Derivatives other than the alcohol include the 6-armamine.

For example, the 10 kDa 6-arm amine (cat # P6AM−10) could be convertedto a 40 kDa 6-arm hexa-amine (with Nektars 5 kDa BocNH-PEG-NHS ester)and then conjugated to a small molecule.

These PEG polymers may be further modified by extending the chains withPEG diamines through an appropriate linker, for example a carbamate(urethane) or a urea.

modified linear PEGs

“Pharmaceutically acceptable salt” refers to salts which retain thebiological effectiveness and properties of the compounds of thisinvention and which are not biologically or otherwise undesirable. Inmany cases, the compounds of this invention are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group.

Examples of suitable amines include, by way of example only,isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine,tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine,purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and thelike. It should also be understood that other carboxylic acidderivatives would be useful in the practice of this invention, forexample, carboxylic acid amides, including carboxamides, lower alkylcarboxamides, dialkyl carboxamides, and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

The term “pharmaceutically-acceptable cation” refers to the cation of apharmaceutically-acceptable salt.

It is understood that in all substituted groups defined herein, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. In such cases, the maximumnumber of such substituents is three. That is to say that each of theabove definitions is constrained by a limitation that, for example,substituted aryl groups are limited to -substituted aryl-(substitutedaryl)-(substituted aryl).

Similarly, it is understood that the above definitions are not intendedto include impermissible substitution patterns (e.g., methyl substitutedwith 5 fluoro groups or a hydroxyl group alpha to ethenylic oracetylenic unsaturation). Such impermissible substitution patterns arewell known to the skilled artisan.

Compound Preparation

The conjugates of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Second Edition, Wiley, New York, 1991, and references citedtherein.

Furthermore, the compounds of this invention will typically contain oneor more chiral centers. Accordingly, if desired, such compounds can beprepared or isolated as pure stereoisomers, ie., as individualenantiomers or diastereomers, or as stereoisomer-enriched mixtures. Allsuch stereoisomers (and enriched mixtures) are included within the scopeof this invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents and the like.

The conjugates of this invention preferably comprise a polymermoiety/optional branched-arm hub molecule containing 2 to about 20substituents of Formula II:

Specifically, the polymer moiety can be bound through a covalent bond tothe Ar¹ substituent, the J substituent, the Ar² substituent and/or inthe T substituent wherein the polymer moiety is either directly attachedor is attached via a linker. In turn, the polymer moiety may optionallybe bound to a branched-arm hub molecule.

In its simplest form, the compounds of this invention are divalentstructures comprising a single polymer moiety having two substituents offormula II bound to both termini. In a representative case using apolymer moiety derived from PEG which is linked to a compound of formulaII by a carbonyl linking group wherein the compound of formula II isrepresented by:

the resulting conjugate can be represented by the following formula:

where p is preferably an integer of from about 100 to 1360.

In one example of a tetravalent form, the conjugate comprises fourpolymer moieties. In a representative case, one terminus of each polymermoiety is attached to a common a branched-arm hub molecule whereas theother terminus is attached to a compound of formula II optionallythrough a linker. Still further and again for illustrative purposes,each polymer moiety is derived from PEG and the common branched-arm hubmolecule is pentaerythritol. In this exemplification, the other terminusof the PEG moiety is linked to a compound of formula II through acarbonyl linking group wherein the compound of formula II is representedby:

the resulting conjugate can be represented by the following formula:

where the aggregate of the four p's is an integer preferably of fromabout 100 to 1360.

The synthetic protocol for forming the conjugates of formula I entailsreaction of a functional group on the polymer moiety with either alinking group or directly with a compound of formula II therebycovalently binding the polymer moiety to the compound of formula II.

Initially, non-PEG substituted compounds of Formula IIb-IIh are wellknown in the art and are exemplified in a number of issued patentsincluding, without limitation, U.S. Pat. Nos. 6,489,300 and 6,436,904both of which are incorporated herein by reference in their entirety.Non-polymer variants of compounds of Formula II include those havingcomplementary functional groups or groups derivatizable to complementaryfunctional groups on one or more of the Ar¹, R, Ar² and T moieties. Forillustrative purposes, compounds having a complementary functional group(—OH) on the Ar² moiety (e.g., tyrosine) are recited below as a suitablestarting point for addition of a polymer moiety to the molecule eitherdirectly or through a linker.

Such compounds can be prepared by first coupling a heterocyclic aminoacid, 1, with an appropriate aryl sulfonyl chloride as illustrated inScheme 1 below:

where R, Ar¹, X, m and n are as defined above.

Specifically, in Scheme 1 above, heterocyclic amino acid, 1, is combinedwith a stoichiometric equivalent or excess amount (preferably from about1.1 to about 2 equivalents) of arylsulfonyl halide, 2, in a suitableinert diluent such as dichloromethane and the like. Generally, thereaction is conducted at a temperature ranging from about −70° C. toabout 40° C. until the reaction is substantially complete, whichtypically occurs within 1 to 24 hours. Preferably, the reaction isconducted in the presence of a suitable base to scavenge the acidgenerated during the reaction. Suitable bases include, by way ofexample, tertiary amines, such as triethylamine, diisopropylethylamine,N-methyl-morpholine and the like. Alternatively, the reaction can beconducted under Schotten-Baumann-type conditions using an aqueous alkalisolution such as an aqueous solution of sodium hydroxide, an aqueousphosphate solution buffered to pH 7.4, and the like. The resultingproduct, 3, can be recovered by conventional methods, such aschromatography, filtration, evaporation, crystallization, and the likeor, alternatively, used in the next step without purification and/orisolation.

Heterocyclic amino acids, 1, employed in the above reaction are eitherknown compounds or compounds that can be prepared from known compoundsby conventional synthetic procedures. Examples of suitable amino acidsfor use in this reaction include, but are not limited to, L-proline,trans-4-hydroxyl-L-proline, cis-4-hydroxyl-L-proline,trans-3-phenyl-L-proline, cis-3-phenyl-L-proline, L-(2-methyl)proline,L-pipecolinic acid, L-azetidine-2-carboxylic acid,L-thiazolidine-4-carboxylic acid,L-(5,5-dimethyl)thiazolidine-4-carboxylic acid,L-thiamorpholine-3-carboxylic acid. If desired, the correspondingcarboxylic acid esters of the amino acids, 1, such as the methyl esters,ethyl esters, t-butyl esters, and the like, can be employed in the abovereaction with the arylsulfonyl chloride. Subsequent hydrolysis of theester group to the carboxylic acid using conventional reagents andconditions, i.e., treatment with an alkali metal hydroxide in an inertdiluent such as methanol/water, then provides the N-sulfonyl amino acid,3.

Similarly, the arylsulfonyl chlorides, 2, employed in the above reactionare either known compounds or compounds that can be prepared from knowncompounds by conventional synthetic procedures. Such compounds aretypically prepared from the corresponding sulfonic acid, i.e., fromcompounds of the formula Ar¹SO₃H where Ar¹ is as defined above, usingphosphorous trichloride and phosphorous pentachloride. This reaction isgenerally conducted by contacting the sulfonic acid with about 2 to 5molar equivalents of phosphorous trichloride and phosphorouspentachloride, either neat or in an inert solvent, such asdichloromethane, at temperature in the range of about 0° C. to about 80°C. for about 1 to about 48 hours to afford the sulfonyl chloride.Alternatively, the arylsulfonyl chlorides, 2, can be prepared from thecorresponding thiol compound, i.e., from compounds of the Ar¹—SH whereAr¹ is as defined herein, by treating the thiol with chlorine (Cl₂) andwater under conventional reaction conditions.

Alternatively, arylsulfonyl chlorides, 2, employed in the above reactionmay be prepared by chlorosulfonylation of substituted benzene orheterocycloalkyl group using Cl—SO₃H.

Examples of arylsulfonyl chlorides suitable for use in this inventioninclude, but are not limited to, benzenesulfonyl chloride,1-naphthalenesulfonyl chloride, 2-naphthalenesulfonyl chloride,p-toluenesulfonyl chloride, o-toluenesulfonyl chloride,4-acetamidobenzenesulfonyl chloride, 4-tert-butylbenzenesulfonylchloride, 4-bromobenzenesulfonyl chloride, 2-carboxybenzenesulfonylchloride, 4-cyanobenzenesulfonyl chloride, 3,4-dichlorobenzenesulfonylchloride, 3,5-dichlorobenzenesulfonyl chloride,3,4-dimethoxybenzenesulfonyl chloride,3,5-ditrifluoromethylbenzenesulfonyl chloride, 4-fluorobenzenesulfonylchloride, 4-methoxybenzenesulfonyl chloride,2-methoxycarbonylbenzenesulfonyl chloride, 4-methylamido-benzenesulfonylchloride, 4-nitrobenzenesulfonyl chloride,4-trifluoromethyl-benzenesulfonyl chloride,4-trifluoromethoxybenzenesulfonyl chloride,2,4,6-trimethylbenzenesulfonyl chloride, 2-thiophenesulfonyl chloride,5-chloro-2-thiophenesulfonyl chloride, 2,5-dichloro-4-thiophenesulfonylchloride, 2-thiazolesulfonyl chloride, 2-methyl-4-thiazolesulfonylchloride, 1-methyl-4-imidazolesulfonyl chloride,1-methyl-4-pyrazolesulfonyl chloride,5-chloro-1,3-dimethyl-4-pyrazolesulfonyl chloride, 3-pyridinesulfonylchloride, 2-pyrimidinesulfonyl chloride and the like. If desired, asulfonyl fluoride, sulfonyl bromide or sulfonic acid anhydride may beused in place of the sulfonyl chloride in the above reaction to form theN-sulfonyl amino acid, 3.

The N-arylsulfonyl amino acid, 3, is then coupled to commerciallyavailable tyrosine esters as shown in Scheme 2 below:

where R, Ar¹, X, m and n are as defined above, R^(a) is hydrogen oralkyl but preferably is an alkyl group such as t-butyl, Z representsoptional substitution on the aryl ring and o is zero, one or two.

This coupling reaction is typically conducted using well-known couplingreagents such as carbodiimides, BOP reagent(benzotriazol-1-yloxy-tris(dimethylamino)-phosphoniumhexafluorophosphonate) and the like. Suitable carbodiimides include, byway of example, dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and the like. Ifdesired, polymer supported forms of carbodiimide coupling reagents mayalso be used including, for example, those described in TetrahedronLetters, 34(48), 7685 (1993). Additionally, well-known couplingpromoters, such as N-hydroxysuccinimide, 1-hydroxybenzotriazole and thelike, may be used to facilitate the coupling reaction.

This coupling reaction is typically conducted by contacting theN-sulfonylamino acid, 3, with about 1 to about 2 equivalents of thecoupling reagent and at least one equivalent, preferably about 1 toabout 1.2 equivalents, of tyrosine derivative, 4, in an inert diluent,such as dichloromethane, chloroform, acetonitrile, tetrahydrofuran,N,N-dimethylformamide and the like. Generally, this reaction isconducted at a temperature ranging from about 0° C. to about 37° C. forabout 12 to about 24 hours. Upon completion of the reaction, thecompound 5 is recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like.

Alternatively, the N-sulfonyl amino acid, 3, can be converted into anacid halide which is then coupled with compound, 4, to provide compound5. The acid halide can be prepared by contacting compound 3 with aninorganic acid halide, such as thionyl chloride, phosphoroustrichloride, phosphorous tribromide or phosphorous pentachloride, orpreferably, with oxalyl chloride under conventional conditions.Generally, this reaction is conducted using about 1 to 5 molarequivalents of the inorganic acid halide or oxalyl chloride, either neator in an inert solvent, such as dichloromethane or carbon tetrachloride,at temperature in the range of about 0° C. to about 80° C. for about 1to about 48 hours. A catalyst, such as DMF, may also be used in thisreaction.

The acid halide of N-sulfonyl amino acid, 3, is then contacted with atleast one equivalent, preferably about 1.1 to about 1.5 equivalents, ofthe tyrosine derivative, 4, in an inert diluent, such asdichloromethane, at a temperature ranging from about −70° C. to about40° C. for about 1 to about 24 hours. Preferably, this reaction isconducted in the presence of a suitable base to scavenge the acidgenerated during the reaction. Suitable bases include, by way ofexample, tertiary amines, such as triethylamine, diisopropylethylamine,N-methylmorpholine and the like. Alternatively, the reaction can beconducted under Schotten-Baumann-type conditions using aqueous alkali,such as sodium hydroxide and the like. Upon completion of the reaction,compound 5 is recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like.

Alternatively, compound 5 can be prepared by first forming a diaminoacid derivative and then coupling the diamino acid to the arylsulfonylhalide, 2, as shown in scheme 3 below:

where R, R^(a), Ar¹, X, Z, m, n and o are as defined above.

The diamino acid, 6, can be readily prepared by coupling amino acid, 1,with amino acid, 4, using conventional amino acid coupling techniquesand reagents, such carbodiimides, BOP reagent and the like, as describedabove. Diamino acid, 6, can then be sulfonated using sulfonyl chloride,2, and using the synthetic procedures described above to providecompound 7.

The tyrosine derivatives, 4, employed in the above reactions are eitherknown compounds or compounds that can be prepared from known compoundsby conventional synthetic procedures. For example, tyrosine derivatives,4, suitable for use in the above reactions include, but are not limitedto, L-tyrosine methyl ester, L-tyrosine t-butyl ester,L-3,5-diiodotyrosine methyl ester, L-3-iodotyrosine methyl ester,β-(4-hydroxy-naphth-1-yl)-L-alanine methyl ester,β-(6-hydroxy-naphth-2-yl)-L-alanine methyl ester, and the like. Ifdesired, of course, other esters or amides of the above-describedcompounds may also be employed.

The N-arylsulfonyl-heterocyclic amino acid-tyrosine derivative, 7, canbe used as a starting point to attach a polymer moiety at the Ar² groupby coupling reactions shown in Schemes 4-14 below which couplingreactions are illustrative only in demonstrating how polymer moietiescan be introduced. In these Schemes, PEG is used as the polymer moietyfor illustrative purposes only. It is understood that other suitablepolymers could be used in place of PEG and that one of ordinary skill inthe art would readily be able to modify the reaction schemes below toincorporate such other polymers. In some cases, the PEG moiety can bedirectly introduced onto the phenoxy group and, in other cases, the PEGmoiety can be introduced by linkage through a linker moiety.

Specifically, Scheme 4 illustrates the following:

wherein Ar¹, R, R^(a), m, n, o, X, and Z are as defined above, Pg is anamine protecting group such as CBZ, Boc, etc. which is preferablyorthogonally removable as compared to the R^(a) carboxyl protectinggroup and p is an integer preferably of from about 100 to 1360.

Specifically, in Scheme 4, compound 7, prepared as above, is combinedwith at least an equivalent and preferably an excess of 4-nitrophenylchloroformate 8, in a suitable solvent such as methylene chloride,chloroform and the like and preferably under an inert atmosphere. Thereaction is preferably conducted at a temperature of from about −40° toabout 0° C. in the presence of a suitable base to scavenge the acidgenerated. Suitable bases include, by way of example, triethylamine,diisopropylethylamine, and the like. After formation of the intermediatemixed carbonate (not shown), at least an approximately equimolar amountof N—Pg piperazine, 8a, is added to the reaction solution. This reactionis allowed to continue at room temperature for about 1 to 24 hours. Uponcompletion of the reaction, compound 9 is recovered by conventionalmethods including neutralization, evaporation, extraction,precipitation, chromatography, filtration, and the like, or,alternatively, is used in the next reaction without purification and/orisolation.

Conventional removal of the protecting group provides for the freepiperazine derivative, 10. Removal is accomplished in accordance withthe blocking group employed. For example, a trifluoromethylcarbonylprotecting group is readily removed via an aqueous solution of potassiumcarbonate. Further, suitable protecting groups for various functionalgroups as well as suitable conditions for protecting and deprotectingparticular functional groups are well known in the art. See, forexample, T. W. Greene and G. M. Wuts, Protecting Groups in OrganicChemistry, Second Edition, Wiley, New York, 1991, and references citedtherein.

The free piperazine derivative, 10, is then combined with anα,ω-dichloroformate polyoxyethylene, compound 11, in a suitable inertdiluent such as methylene chloride, chloroform, and the like andpreferably under an inert atmosphere. Typically, at least 2 equivalentsand preferably from about 2.5 to 10 equivalents of compound 10 perchloroformate entity are employed in combination with compound 11. Thereaction is optionally conducted in the presence of a catalytic amountof DMAP and a base to scavenge the acid generated during reaction. Thereaction is continued under ambient conditions until substantiallycomplete which typically occurs within 4 to 24 hours. When R^(a) isalkyl, subsequent hydrolysis of the ester derivative provides for thefree carboxyl group or a salt thereof. The resulting dimer, 12, isrecovered by conventional procedures such as neutralization,evaporation, extraction, precipitation, chromatography, filtration, andthe like.

The α,ω-dichloroformate polyoxyethylene, compound 11, is readilyprepared from commercially available polyoxyethylene by reaction with anexcess of phosgene, typically from at least 2 to about 20 equivalents,in a suitable inert solvent such as methylene chloride, chloroform andthe like. The reaction is preferably conducted under an inert atmosphereat ambient conditions until the reaction is substantially complete whichtypically occurs in from about 2 to 24 hours. Afterwards, the resultingα,ω-dichloroformate polyoxyethylene, compound 11, is recovered byconvention procedures such as neutralization, evaporation, extraction,precipitation, chromatography, filtration, and the like.

A specific example of this reaction scheme up to formation of thepiperazine derivative 28 is illustrated in Scheme 5 below:

Specifically, commercially available 3-pyridinesulfonic acid, 21, isconverted under conventional conditions to the corresponding sulfonylchloride, 22, by contact with POCl₃/PCl₅ using conditions well known inthe art. Coupling of sulfonyl chloride, 22, with commercially availableS-5,5-dimethylthiazolidine-4-carboxylic acid, 23, is accomplished underconventional conditions preferably in the presence of a phosphate buffer(pH 7.4) using an excess of sulfonyl chloride. The reaction ispreferably conducted at a temperature of from about −10 to 20° C. untilthe reaction is substantially complete, which typically occurs within0.5 to 5 hours. The resulting product, 24, can be recovered byconventional methods, such as chromatography, filtration, evaporation,crystallization, and the like or, alternatively, used in the next stepwithout purification and/or isolation.

The N-pyridinyl sulfonyl-5,5-dimethylthiazolidine-4-carboxylic acidcompound, 23, is next coupled to t-butyl tyrosine using conventionalamino acid coupling conditions. Specifically, this coupling reaction isconducted using well known coupling reagents such as1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC),1-hydroxy-benzotriazole (HOBt) and N-methylmorpholine to facilitate thecoupling reaction.

This coupling reaction is typically conducted by contacting theN-sulfonylamino acid, 23, with about 1 to about 2 equivalents of thecoupling reagent and at least one equivalent, preferably about 1 toabout 1.2 equivalents, of tyrosine t-butyl ester in an inert diluent,such as dichloromethane, chloroform, acetonitrile, tetrahydrofuran,N,N-dimethylformamide and the like. Generally, this reaction isconducted at a temperature ranging from about 0° C. to about 22° C. forabout 12 to about 24 hours. Upon completion of the reaction, thecompound 24 is recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like or, alternatively, is employed in the next stepwithout purification and/or isolation.

Separately, mono-N-Boc-piperazine, 25, is converted to the correspondingcarbamyl chloride, 26, by reaction with phosgene in the manner describedabove. Upon completion of the reaction, the compound 26 is recovered byconventional methods including neutralization, evaporation, extraction,precipitation, chromatography, filtration, and the like or,alternatively, is employed in the next step without purification and/orisolation.

Coupling of compound 24 with compound 26 to provide for compound 27proceeds under conventional conditions in an inert diluent such asdichloromethane, with a catalytic amount of DMAP and preferably in thepresence of a base to scavenge the acid generate. The reaction is run ata temperature of about −20 to about 22° C. for about 2 to about 24hours. Upon completion of the reaction, compound 27 is recovered byconventional methods including neutralization, evaporation, extraction,precipitation, chromatography, filtration, and the like or,alternatively, is employed in the next step without purification and/orisolation.

Removal of both the amino Boc protecting group and the t-butyl esterproceeds in the presence of trifluoroacetic acid to provide for compound28 which can be recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like.

Scheme 6 below illustrates the preparation of a piperazine compoundorthogonally protected on one of the amine groups relative to thecarboxyl protecting group found on the phenylalanine compound such thatafter coupling, the piperazine protecting group can be removeddifferentially from that of the carboxyl protecting group. Suchorthogonal protection is necessary if subsequent reactions on theresulting compound require a carboxyl protecting group to avoidundesired side reactions.

Specifically, in Scheme 6, compound 24 is prepared in the mannerdescribed above. N-t-Boc-piperazine, 25, is conventionally converted toN-t-Boc-N′-trifluoromethyl-carbonylpiperazine, 29, by contact with anexcess of trifluoroacetic anhydride in the presence of a suitable aminesuch as triethylamine to scavenge the acid generated during reaction ina suitable solvent such as dichloromethane. Generally, this reaction isconducted at a temperature ranging from about −20° C. to about 22° C.for about 1 to about 24 hours. Upon completion of the reaction, compound29 can be recovered by conventional methods including neutralization,evaporation, extraction, precipitation, chromatography, filtration, andthe like or, alternatively and preferably, is employed in the next stepwithout purification and/or isolation.

In turn, removal of the t-Boc protecting group on theN-t-Boc-N′-trifluoromethyl-carbonylpiperazine, 29, proceeds underconventional conditions using gaseous HCl bubbled through an inertsolvent such as methylene chloride, EtOAc, EtO₂, and the like underambient conditions to provide for the hydrochloride salt ofN′-trifluoromethylcarbonylpiperazine, 30. Generally, this reaction isconducted at a temperature ranging from about −20° C. to about 22° C.for about 0.5 to about 4 hours. Upon completion of the reaction,compound 30 can be recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like or, alternatively and preferably, is employedin the next step without purification and/or isolation.

Conversion of N′-trifluoromethylcarbonylpiperazine, 30, to theN-carbamyl chloride derivative, 31, conventionally proceeds by contactwith phosgene in the manner described above. Upon completion of thereaction, compound 31 can be recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like or, alternatively and preferably, is employedin the next step without purification and/or isolation.

Compounds 31 and 24 are coupled under conditions similar to thosedescribed above to provide for compound 32 which is orthogonallyprotected at the amino moiety of the piperazine group as well as thecarboxyl moiety of the phenylalanine group. Selective removal of thetrifluoromethylcarbonyl amino protecting group proceeds underconventional conditions using an aqueous solution of potassium carbonateto provide for compound 33.

Scheme 7 below illustrates modification of the polymer moiety prior tocovalently binding the compound of formula II. For illustrative purposesonly, the polymer moiety is a tetravalent PEG bound to apentaerythritol. Scheme 7 illustrates that the length of the polymermoiety can be readily adjusted by conventional chemistry to provide foroptimal lengths.

where the aggregate of the four r's and s's is an integer preferablyfrom about 100 to 1360.

Specifically, commercially available tetra-pegylated pentaerythritol,compound 1034, (e.g., a compound having a total molecular weight ofapproximately 20 kD and available from Sun Bio, Orinda, Calif., USA, ascatalog no. P40H-20), is reacted with an excess of phosgene, typicallyfrom at least 4 to about 40 equivalents, in a suitable inert solventsuch as methylene chloride, chloroform and the like. The reaction ispreferably conducted under an inert atmosphere at ambient conditionsuntil the reaction is substantially complete which typically occurs infrom about 2 to 24 hours. Afterwards, the resulting tetrachloroformatepolyoxyethylene, compound 35, is recovered by convention procedures suchas neutralization, evaporation, extraction, precipitation,chromatography, filtration, and the like or is used in the next reactionstep without purification and/or isolation.

Tetrachloroformate, compound 35, is then combined with an excess(typically 2.5 to 10 equivalents per chloroformate entity) of anα,ω-diaminopolyoxyethylene compound (e.g., a compound having a molecularweight of approximately 6 kD and available from Sun Bio, as catalog no.P2AM−6), under conventional conditions in an inert diluent such asdichloromethane, optionally in the presense of a catalytic amount ofDMAP and a base to scavenge the acid generate. The reaction is typicallyconducted at a temperature of about −20 to about 22° C. for about 2 toabout 24 hours or until substantial completion of the reaction Uponcompletion, compound 36 is recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like or, alternatively, is employed in the next stepwithout purification and/or isolation.

When the specific tetra-pegylated pentaerythritol from Sun Bio and thediamine from Sun Bio are employed, the resuting product, compound 36,has a molecular weight of approximately 45 kD.α,ω-Diaminopolyoxyethylenes are commercially available under thetradename Jeffamines® and typically have molecular weights of up to10,000 or higher.

It is understood that a mono-amino protected α,ω-diaminopolyoxyethylenemay be used in Scheme 7 in order to minimize cross-linking as well ascyclization. Upon reaction completion, the mono-amino protecting groupis removed by conventional means well known in the art.

Scheme 8 illustrates a second route for derivatization to provide forpolymer substitution. In this scheme, the amino moiety of the piperazinegroup is employed as a complementary functional group to an in situformed activated carboxyl groups of an α,ω-dicarboxylic acid polymer.Again for the sake of illustration only, the α,ω-dicarboxylic acidpolymer is an α,ω-dicarboxylic acid polyoxyethylene. In this embodiment,the dicarboxyl-PEG compound is represented by the formulaHOOCCH₂(OCH₂CH₂)_(p)OCH₂COOH where p is as defined above and theresulting linker to the PEG group is represented by —C(O)CH₂—.

Specifically, in Scheme 8, an excess of compound 33 (e.g., 2.5 to 10equivalents of compound 33 per carboxyl group), prepared as above, isadded to the dicarboxyl-PEG compound which is converted in situ to anactivated ester (not shown) by contact with at least two equivalents andpreferably an excess of HATU[O-(7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate] in the presence of a suitable amine such astriethylamine. Coupling of the dicarboxyl-PEG compound to compound 33preferably proceeds at a temperature of from about 0 to about 22° C. forabout 2 to about 24 hours. Upon completion of the reaction, the compound39 is recovered by conventional methods including neutralization,evaporation, extraction, precipitation, chromatography, filtration, andthe like or, alternatively, is employed in the next step withoutpurification and/or isolation.

Conventional removal of the t-butyl carboxyl protecting group with anexcess of formic acid provides for a compound of Formula IA of thisinvention.

Scheme 9 illustrates still another route for derivatization to providefor polymer addition to compound A. In this scheme, the amino moiety ofthe piperazine group is employed as a complementary functional group toan in situ formed chloroformate of a polymer comprsing an α,ω-diol.Again for illustrative purposes, the polymer comprising an α,ω-diol isPEG which is represented by the formula HOCH₂CH₂(OCH₂CH₂)_(p)OH where pis as defined above and the resulting linker is represented by —C(O)—.

Specifically, in Scheme 9, the hydroxyl group of a commerciallyavailable dihydroxy PEG, 42, is converted to the correspondingchloroformate, 37, by reaction with phosgene in toluene (Fluka), indichloromethane. The product is isolated by evaporation and is employedin the next step without further purification.

An excess of compound 33 (e.g, 2.5 to 10 equivalent of compound 33 perchloroformate entity) is contacted with dichloroformate, compound 43,prepared as above, in the presence of a suitable base such astriethylamine to scavenge the acid generated. Coupling of thedichloroformate-PEG compound to compound 33 preferably proceeds at atemperature of from about 0 to about 22° C. for about 2 to about 4hours. Upon completion of the reaction, the compound 44 is recovered byconventional methods including neutralization, evaporation, extraction,precipitation, chromatography, filtration, and the like or,alternatively, is employed in the next step without purification and/orisolation.

Conventional removal of the t-butyl carboxyl protecting group with anexcess of formic acid provides for a compound of Formula I of thisinvention.

The reactions depicted in Schemes 8 and 9 are simultaneously conductedat either end of the dicarboxylic acid (Scheme 8) or the dichloroformate(Scheme 9) thereby providing a one pot synthesis of a homomeric divalentor higher multivalent conjugate. It is understood, however, that thesereactions can be conducted sequentially by use of protecting groups.

In the case of a dicarboxylic acid, one of the carboxyl groups can beprotected while the other undergoes coupling to the amino group of thepiperazine. Upon completion, the protecting group can be removed andthen reacted with either the same or preferably a different compound Ato provide for a heterodivalent structure. Still further,heterotrivalent, heterotetravalent and higher heteromultivalentstructures can be prepared by use of orthogonal protecting groups on thecarboxylic functionality. In the case of a diol (Scheme 9), one of thehdyroxyl groups can be protected while the other undergoes reaction withphosgene to form a chloroformate for subsequent addition to the aminogroup of the piperazine. Upon completion, the protecting group can beremoved and then reacted with phosgene and subsequently with either thesame or preferably a different compound A to provide for aheterodivalent structure. Still further, heterotrivalent,heterotetravalent and higher heteromultivalent structures can beprepared by use of orthogonal protecting groups on the alcoholfunctionality.

Scheme 10 illustrates the synthesis of N-carbamyl chloride andisocyanate intermediates useful for subsequent polymer addition. In thisscheme, the amino moiety of the piperazine group is derivatized forsubsequent polymer addition.

Specifically, in Scheme 10, conversion of the amino moiety of thepiperazine group of compound 33, to the corresponding N-carbamylchloride, compound 33a, proceeds by contact with an excess of phosgenein the presence of a suitable base such as sodium bicarbonate toscavenge the acid generated during reaction. Upon completion of thereaction, compound 33a can be recovered by conventional methods such asneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like or, alternatively and preferably is employed inthe next (illustrated in Scheme 11) without purification and/orisolation.

Alternatively, the amino moiety of the piperazine group of compound 33can be converted to the corresponding amide, compound 45, by reactionwith at least an equivalent and preferably an excess of 4-nitrobenzoylchloride in the presence of a base such as pyridine (which can also actas a solvent) to scavenge the acid generated during reaction. Thereaction preferably proceeds at a temperature of from about 0 to about22° C. for about 1 to about 24 hours. Upon completion of the reaction,compound 45 is recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like or, alternatively, is employed in the next stepwithout purification and/or isolation.

Subsequent reduction of the para-nitro substituent of the phenyl groupprovides for the amine substituent in compound 46. Reduction isconventionally conducted using palladium/carbon under a hydrogenatmosphere typically at elevated pressures in a suitable diluent such asmethanol. The reaction proceeds until substantial completion whichtypically occurs within about 24 to about 72 hours. During the reaction,additional catalyst is added as required to affect reaction completion.Upon completion of the reaction, the compound 46 is recovered byconventional methods including neutralization, evaporation, extraction,precipitation, chromatography, filtration, and the like or,alternatively, is employed in the next step without purification and/orisolation.

Conversion of the para-amino substituent of the phenyl group of compound46 to the corresponding isocyanate, 47, occurs by reaction with anexcess of phosgene in the presence of a suitable base such as sodiumbicarbonate which scavenges the acid generated. The reaction proceedsuntil substantial completion which typically occurs within about 0.5 toabout 5 hours at about 0° C. to about 22° C. Upon completion of thereaction, the compound 47 is recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like or, alternatively, is employed in the next stepwithout purification and/or isolation.

Scheme 11 illustrates still a further route for derivatization toprovide for polymer substitution. In this scheme, the carbamyl chloridemoiety of the piperazine group of compound 33a is employed as acomplementary functional group to form a carbamate or urea bond. Forillustrative purposes only, the polymer employed is an α,ω-diol ordiamine of a PEG and is represented by the formulaHQCH₂CH₂(OCH₂CH₂)_(p)QH where Q is NH or O.

Specifically, in Scheme 11, an excess (e.g., 2.5 to 10 equivalents ofcarbamyl chloride per each HQ moiety) of compound 33a, is contacted inan inert solvent such as dichloromethane with a suitable dihydroxy- ordiamino-PEG compound preferably in the presence of a suitable base suchas triethylamine and/or catalytic amounts of 4-N,N-dimethylaminopyridine(DMAP). The reaction proceeds until substantial completion whichtypically occurs within about 4 to about 48 hours. Upon completion ofthe reaction, the compound 48 is recovered by conventional methodsincluding neutralization, evaporation, extraction, precipitation,chromatography, filtration, and the like or, alternatively, is employedin the next step without purification and/or isolation.

When Q is a hydroxyl group, the product contains a carbamatefunctionality covalently linking the PEG group to the VLA-4 antagonistthrough a linker represented by —C(O)—. When Q is an amino group, theproduct contains a urea functionality covalently linking the PEG groupto the VLA-4 antagonist through a linker represented by—C(O)-Conventional removal of the t-butyl carboxyl protecting group withan excess of formic acid provides for a compound of this invention.

Scheme 12 illustrates yet another route for derivatization to providefor polymer substitution. In this scheme, the isocyanate of compound 47is employed as a complementary functional group to form a carbamate orurea bond. For illustrative purposes only, the polymer employed is anα,ω-diol or diamine of a PEG and is represented by the formulaHQCH₂CH₂(OCH₂CH₂)_(p)QH where Q is NH or O.

Specifically, in Scheme 12, an excess of isocyanate 47 (e.g., 2.5 to 10equivalents of isocyanate 47 per each HQ moiety) is contacted with asuitable dihydroxy- or diamino-PEG compound in a suitable inert diluentsuch as dichloromethane or toluene. The reaction is preferablymaintained at a temperature of from about 0° to about 105° C. untilsubstantial completion which typically occurs within about 1 to about 24hours. Upon completion of the reaction, compound 49 is recovered byconventional methods including neutralization, evaporation, extraction,precipitation, chromatography, filtration, and the like or,alternatively, is employed in the next step without purification and/orisolation.

When Q is a hydroxyl group, the resulting product contains a carbamatefunctionality covalently linking the PEG group to the VLA-4 antagonistthrough a —C(O)— linking group. When Q is an amino group, the resultingproduct contains a urea functionality covalently linking the PEG groupto the VLA-4 antagonist through a —C(O)— linking group.

Conventional removal of the t-butyl carboxyl protecting group with anexcess of formic acid provides for a mono-PEG compound, 47, of Formula Iof this invention.

The reactions depicted in Schemes 11 and 12 are simultaneously conductedat both ends of the polymer (for dimer formation) thereby providing aone pot synthesis of a homomeric divalent or higher multivalentconjugate. It is understood, however, that these reactions can beconducted sequentially by use of protecting groups.

In the case of a diamine, one of the amine groups can be protected whilethe other undergoes coupling to either the carbamyl chloride of compound33a or the isocyanate of compound 47. Upon completion, the protectinggroup can be removed and then reacted with either the same or preferablya different compound A to provide for a heterodivalent structure. Stillfurther, heterotrivalent, heterotetravalent and higher heteromultivalentstructures can be prepared by use of orthogonal protecting groups on oneor more of the amine functionalities.

In the case of a diol, one of the hydroxyl groups can be protected whilethe other undergoes coupling to either the carbamyl chloride of compound33a or the isocyanate of compound 47. Upon completion, the protectinggroup can be removed and then reacted with either the same or preferablya different compound A to provide for a heterodivalent structure. Stillfurther, heterotrivalent, heterotetravalent and higher heteromultivalentstructures can be prepared by use of orthogonal protecting groups on oneor more of the hydroxyl functionalities.

In the Schemes above, amine moieties located on other portions of themolecule can be employed in the manner described above to covalentlylink a polymer group to the molecule. For example, amines located onAr¹, on the heterocyclic amino acid or on Ar² can be similarlyderivatized to provide for PEG substitution. The amine moieties can beincluded in these substituents during synthesis and appropriatelyprotected as necessary. Alternatively, amine precursors can be employed.For example, as shown in Scheme 10, reduction of a nitro group providesfor the corresponding amine. Similarly, reduction of a cyano groupprovides for a H₂NCH₂— group. Nitro and cyano substituted Ar¹ groups areprovided in U.S. Pat. No. 6,489,300 as is an amino substituted Ar¹group.

Further, the amino substitution can be incorporated into theheterocyclic amino acid functionality and then derivatized to include apolymer moiety. For example, the heterocyclic amino acid functionalitycan be 2-carboxylpiperazine depicted in U.S. Pat. No. 6,489,300.Alternatively, commercially available 3- or 4-hydroxyproline can beoxidized to the corresponding ketone and then reductively aminated withammonia in the presence of sodium cyanoborohydride to form thecorresponding amine moiety. Still further, 4-cyanoproline can be reducedto provide for a substituted alkyl group of the formula —CH₂NH₂ whichcan be derivatized through the amine.

Still further, the amine moiety can be incorporated into the Ar²functionality. Preferably, the amine moiety is present as an amineprecursor such as a nitro or cyano group bound to Ar².

In the schemes above, the reactions of the amine with a complementaryfunctional group can be reversed such that the carboxyl or hydroxylgroup is on the VLA-4 antagonist of Formula II (without any polymersubstituents) and the amine group could be part of the polymer moiety.In such cases, the amine group, preferably terminating the polymermoiety, can be converted to an isocyanate, using phosgene and Et₃N, andreacted with the hydroxyl group to form a carbamate as illustrated inScheme 13 below:

Specifically, an excess of compound 50 described in U.S. Pat. No.6,489,300, is contacted with in the manner described above to providefor the corresponding carbamate, 51. Preferably, from about 2.5 to 10equivalents of compound 50 per each isocyanate moiety is employed.Deprotection, as described above, then provides for the correspondingdiacid (not shown).

Alternatively, in Scheme 13, the hydroxyl functionality can be reactedwith phosgene to provide for the chlorocarbonyloxy derivative whichreacts with an amine group of a diamine compound to provide for thecarbamate.

Carboxyl functionality, for example on the Ar¹ moiety, can be convertedto the corresponding amide by reaction with a di- or higher-aminopolymerin the manner described above in Scheme 8. Alteratively, Scheme 14 belowillustrates one method for the generation of an amine functionality fromthe corresponding cyano group on the Ar¹ moiety.

Specifically, in Scheme 14, known compound 52, described in U.S. Pat.No. 6,489,300, is t-butyl protected under convention conditions toprovide the cyano compound 53 which is hydrogenated under conventionalconditions to provide the aminomethyl compound 54. The aminomethyl groupof compound 54 is available for coupling of a polymer moiety thereto inone on any of the schemes illustrated above.

Scheme 15 below illustrates an alternative synthesis of3-aminopyrrolidinyl derivatives useful for coupling a polymer moietythereto in any one of the schemes illustrated above.

Using conventional methods, commercially available cis-4-hydroxyL-proline, 57, is treated with methanolic hydrogen chloride for severalhours at reflux, followed by evaporation, and the so generated methylester hydrochloride is treated with excess tosyl chloride in pyridinefor two days at room temperature, giving the product, 58. Compound 58 isisolated by neutralizing the pyridine using weak aqueous acid andextracting the product with an organic solvent such as EtOAc. Theproduct 58 may be purified by crystallization, flash chromatography, ormore preferably be used in subsequent steps without purification.

Reaction of 58 with a saturated solution of excess sodium azide in DMFat room temperature for 15 days affords compound 59. Compound 59 isisolated by dilution of the reaction mixture with water, followed byextraction with an organic solvent such as EtOAc. The product 59 may bepurified by crystallization, flash chromatography, or more preferably beused in subsequent steps without purification.

Compound 59 is treated with sodium hydroxide, in a mixture of water andmethanol, thus hydrolyzing the methyl ester and generating a carboxylicacid, which is isolated by acidification and extraction with an organicsolvent such as EtOAc. The carboxylic acid is treated with L-tyrosinet-butyl ester [H-Tyr(H)-OtBu], EDAC, HOBt, and Et3N in DMF, generating adipeptide, which is isolated by dilution with water and extraction withan organic solvent such as EtOAc. The dipeptide is treated withCICONMe2, Et3N, and DMAP in DCM at reflux for 24 hours, generating thecarbamate, 60, which is isolated by dilution with EtOAc, sequentialwashing with weak aqueous acid and base, and then evaporation. Compound60 is rigorously purified by flash chromatography.

Finally, compound 61 is prepared by shaking of a solution of 60 inmethanol, with a Pd/C catalyst under an atmosphere of hydrogen. Theproduct, 61, is isolated by removal of the catalyst by filtration andevaporation.

Other methods for coupling of a compound of formula II with a polymer(optionally bound to a a branched-arm hub molecule) are well known inthe art.

Other polymers suitable for conjugation to a compound of formula IIinclude, without limitation, polyvinylpyrrolidone (PVP), polyacrylamide(PAAm), polydimethylacrylamide (PDAAm), polyvinyl alcohol (PVA),dextran, poly (L-glutamic acid) (PGA), styrene maleic anhydride (SMA),poly-N-(2-hydroxypropyl) methacrylamide (HPMA), polydivinylether maleicanhydride (DIVEMA). By way of example, PVP, PAAm and PDAAm may befunctionalized by introduction of co-monomers during radicalpolymerization. PVA and dextran each contain primary hydroxyl (OH)groups suitable for conjugation. Methods for synthesis of thesebiopolymers and for conjugating them to biological materials are wellknown in the art (see, for example, published U.S. Patent Application20040043030; U.S. Pat. No. 5,177,059; U.S. Pat. No. 6,716,821; U.S. Pat.No. 5,824,701; U.S. Pat. No. 6,664,331; U.S. Pat. No. 5,880,131; Kameda,Y. et al., Biomaterials 25: 3259-3266, 2004; Thanou, M. et al, CurrentOpinion in Investigational Drugs 4(6): 701-709, 2003; Veronese, F. M.,et al., II Farmaco 54: 497-516,1999, all of which are incorporatedherein in their entireties).

Pharmaceutical Formulations

When employed as pharmaceuticals, the conjugates of this invention areusually administered in the form of pharmaceutical compositions. Theseconjugates can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular,sublingual, ophthalmic, or inhalation including administration by nasalor oral inhalation. Preferred administration routes includesubcutaneous, intravenous, and inhalation. Such compositions areprepared in a manner well known in the pharmaceutical art and compriseat least one conjugate.

The invention also provides pharmaceutical compositions comprising aconjugate according to the invention, e.g., a conjugate of Formula I, incombination with a separate compound which is an α₄β₇ inhibitor. Suchcompositions also comprise a pharmaceutically acceptable carrier orexcipient and may be administered as discussed elsewhere herein.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the conjugate of formula Itogether with pharmaceutically acceptable carriers. In making thecompositions of this invention, the active ingredient is usually mixedwith an excipient, diluted by an excipient or enclosed within such acarrier which can be in, sterile injectable solutions, and sterilepackaged powders. For subcutaneous administration, a simple carrier maycomprise a sterile solution of water, Na2HPO4, NaH2PO4, and NaCl, inproportions that provide an isotonic and physiologically acceptable pH,known as PBS or phosphate-buffered saline. Another option is toadminister the compounds in sterile isotonic saline adjusted tophysiological pH if needed. Other options are known to those of skill inthe art and include mixed solvent systems that can affect the rate ofabsorption and total exposure. These options include mixed solventsystems containing glycerin, Polyethylene glycol 400, and cottonseedoil. Also of potential use are ethanol, N,N′-dimethylacetamide,propylene glycol and benzyl alcohol all of which may be used tomanipulate permeability enhancement and hypertonicity.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

Administration of therapeutic agents by subcutaneous or intravenousformulation is well known in the pharmaceutical industry. A subcutaneousor intravenous formulation should possess certain qualities aside frombeing just a composition in which the therapeutic agent is soluble. Forexample, the formulation should promote the overall stability of theactive ingredient(s), also, the manufacture of the formulation should becost effective. All of these factors ultimately determine the overallsuccess and usefulness of an intravenous formulation.

Other accessory additives that may be included in pharmaceuticalformulations of compounds of the present invention as follow: solvents:ethanol, glycerol, propylene glycol; stabilizers: EDTA (ethylene diaminetetraacetic acid), citric acid; antimicrobial preservatives: benzylalcohol, methyl paraben, propyl paraben; buffering agents: citricacid/sodium citrate, potassium hydrogen tartrate, sodium hydrogentartrate, acetic acid/sodium acetate, maleic acid/sodium maleate, sodiumhydrogen phthalate, phosphoric acid/potassium dihydrogen phosphate,phosphoric acid/disodium hydrogen phosphate; and tonicity modifiers:sodium chloride, mannitol, dextrose.

The presence of a buffer is necessary to maintain the aqueous pH in therange of from about 4 to about 8 and more preferably in a range of fromabout 4 to about 6. The buffer system is generally a mixture of a weakacid and a soluble salt thereof, e.g., sodium citrate/citric acid; orthe monocation or dication salt of a dibasic acid, e.g., potassiumhydrogen tartrate; sodium hydrogen tartrate, phosphoric acid/potassiumdihydrogen phosphate, and phosphoric acid/disodium hydrogen phosphate.

The amount of buffer system used is dependent on (1) the desired pH; and(2) the amount of drug. Generally, the amount of buffer used is in a0.5:1 to 50:1 mole ratio of buffer:alendronate (where the moles ofbuffer are taken as the combined moles of the buffer ingredients, e.g.,sodium citrate and citric acid) of formulation to maintain a pH in therange of 4 to 8 and generally, a 1:1 to 10:1 mole ratio of buffer(combined) to drug present is used.

A useful buffer in the invention is sodium citrate/citric acid in therange of 5 to 50 mg per ml of sodium citrate to 1 to 15 mg per ml ofcitric acid, sufficient to maintain an aqueous pH of 4-6 of thecomposition.

The buffer agent may also be present to prevent the precipitation of thedrug through soluble metal complex formation with dissolved metal ions,e.g., Ca, Mg, Fe, Al, Ba, which may leach out of glass containers orrubber stoppers or be present in ordinary tap water. The agent may actas a competitive complexing agent with the drug and produce a solublemetal complex leading to the presence of undesirable particulates.

In addition, the presence of an agent, e.g., sodium chloride in anamount of about of 1-8 mg/ml, to adjust the tonicity to the same valueof human blood may be required to avoid the swelling or shrinkage oferythrocytes upon administration of the intravenous formulation leadingto undesirable side effects such as nausea or diarrhea and possibly toassociated blood disorders. In general, the tonicity of the formulationmatches that of human blood which is in the range of 282 to 288 mOsm/kg,and in general is 285 mOsm/kg, which is equivalent to the osmoticpressure corresponding to a 0.9% solution of sodium chloride.

The intravenous formulation can be administered by direct intravenousinjection, i.v. bolus, or can be administered by infusion by addition toan appropriate infusion solution such as 0.9% sodium chloride injectionor other compatible infusion solution.

The compositions may be formulated in a unit dosage form, each dosagecontaining from about 5 to about 100 mg, more usually about 10 to about30 mg, of the active ingredient. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient.

The conjugate is effective over a wide dosage range and is generallyadministered in a pharmaceutically effective amount. It, will beunderstood, however, that the amount of the conjugate actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insulation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be breathed directly from thenebulizing device or the nebulizing device may be attached to a facemasks tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner. For inhalation or insufflation administration,it is preferred that the total molecular weight of the conugate isbetween about 10,000 Daltons and 70,000 Daltons, more preferably betweenabout 20,000 Daltonsand 45,000 Daltons.

Polymer Conjuqates

Compounds of this invention as formulated and administered are polymerconjugates. Polymer conjugates are anticipated to provide benefits overnon-conjugated polymers, such as improved solubility and in vivostability.

As such, single polymer molecule may be employed for conjugation withthe compounds of the present invention, although it is also contemplatedthat more than one polymer molecule can be attached as well, typicallythrough a carrier. The conjugated compounds of the present invention mayfind utility in both in vivo as well as non-in vivo applications.Additionally, it will be recognized that the conjugating polymer mayutilize any other groups, moieties, or other conjugated species, asappropriate to the end use application. As an example, it may beadvantageous in some applications to functionalize the polymer to renderit reactive and enable it to conjugate to a compound of formula II andto enhance various properties or characteristics of the overallconjugated material. Accordingly, the polymer may contain anyfunctionality, repeating groups, linkages, or other constituentstructures which do not preclude the efficacy of the conjugatedcompounds of the present invention for its intended purpose.

Illustrative polymers that are usefully employed to achieve thesedesirable characteristics are described supra, as well as in PCT WO01/54690 (to Zheng et al.) incorporated by reference herein in itsentirety. The polymer may be coupled to the compounds of the presentinvention (preferably via a linker moiety) to form stable bonds that arenot significantly cleavable by human enzymes. Generally, for a bond tobe not ‘significantly’ cleavable requires that no more than about 20% ofthe bonds connecting the polymer and the compounds of the presentinvention to which the polymer is linked, are cleaved within a 24 hourperiod, as measured by standard techniques in the art including, but notlimited to, high pressure liquid chromatography (HPLC).

Generally, the compounds of this invention contain at least about 2compounds of formula II bound to a polymer. The final amount is abalance between maximizing the extent of the reaction while minimizingnon-specific modifications of the product and, at the same time,defining chemistries that will maintain optimum activity, while at thesame time optimizing the half-life of the compounds of the presentinvention. Preferably, at least about 50% of the biological activity ofthe compounds of the present invention is retained, and most preferably100% is retained.

As noted above in the preferred practice of the present invention,polyalkylene glycol residues of C₂-C₄ alkyl polyalkylene glycols,preferably polyethylene glycol (PEG), or poly(oxy)alkylene glycolresidues of such glycols are advantageously incorporated in the polymersystems of interest. Thus, the polymer to which the compounds of thepresent invention are attached may be a homopolymer of polyethyleneglycol (PEG) or is a polyoxyethylated polyol, provided in all cases thatthe polymer is soluble in water at room temperature. Non-limitingexamples of such polymers include polyalkylene oxide homopolymers suchas PEG or polypropylene glycols, polyoxyethylenated glycols, copolymersthereof and block copolymers thereof, provided that the water solubilityof the block copolymer is maintained.

Examples of polyoxyethylated polyols include, but are not limited to,polyoxyethylated glycerol, polyoxyethylated sorbitol, polyoxyethylatedglucose, or the like. The glycerol backbone of polyoxyethylated glycerolis the same backbone occurring naturally in, for example, animals andhumans in mono-, di-, and triglycerides. Therefore, this branching wouldnot necessarily be seen as a foreign agent in the body.

Those of ordinary skill in the art will recognize that the foregoinglist is merely illustrative and that all polymer materials having thequalities described herein are contemplated. The polymer need not haveany particular molecular weight, but it is preferred that the molecularweight be between about 100 and 100,000, preferably from about 10,000 to80,000; more preferably from about 20,000 to about 70,000. Inparticular, sizes of 20,000 or more are most effective at preventingloss of the product due to filtration in the kidneys.

By PEG derivative is meant a polyethylene glycol polymer in which one orboth of the terminal hydroxyl groups found in polyethylene glycol itselfhas been modified. Examples of suitable modifications include replacingone or both hydroxyl group(s) with alternative functional groups, whichmay be protected or unprotected, with low molecular weight ligands, orwith another macromolecule or polymer. Modification of the terminalhydroxyl groups in the polyethylene glycol may be achieved by reactingthe polyethylene glycol with compounds comprising complementary reactivefunctional groups, including functional groups which are able to undergoa reaction with the hydroxyl groups in polyethylene glycol. The PEGderivatives of the compounds of this invention may contain one or morepolyethylene glycol (PEG) substituents covalently attached thereto by alinking group.

The following formulation examples illustrate the pharmaceuticalcompositions of the present invention.

FORMULATION EXAMPLE 1

Hard gelatin capsules containing the following ingredients are prepared:Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

FORMULATION EXAMPLE 2

A tablet formula is prepared using the ingredients below: QuantityIngredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

FORMULATION EXAMPLE 3

A dry powder inhaler formulation is prepared containing the followingcomponents: Ingredient Weight % Active Ingredient 5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling appliance.

FORMULATION EXAMPLE 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows: Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mgStarch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0 mg (as 10% solution in sterile water) Sodium carboxymethyl starch 4.5 mg Magnesium stearate  0.5 mg Talc  1.0 mg Total  120 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° C. to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

FORMULATION EXAMPLE 5

Capsules, each containing 40 mg of medicament are made as follows:Quantity Ingredient (mg/capsule) Active Ingredient  40.0 mg Starch 109.0mg Magnesium stearate  1.0 mg Total 150.0 mg

The active ingredient, starch and magnesium stearate are blended, passedthrough a No. 20 mesh U.S. sieve, and filled into hard gelatin capsulesin 150 mg quantities.

FORMULATION EXAMPLE 6

Suppositories, each containing 25 mg of active ingredient are made asfollows: Ingredient Amount Active Ingredient   25 mg Saturated fattyacid glycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

FORMULATION EXAMPLE 7

Suspensions, each containing 50 mg of medicament per 5.0 ml dose aremade as follows: Ingredient Amount Active Ingredient 50.0 mg Xanthan gum4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose(89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Colorq.v. Purified water to 5.0 ml

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

FORMULATION EXAMPLE 8

Quantity Ingredient (mg/capsule) Active Ingredient  15.0 mg Starch 407.0mg Magnesium stearate  3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 425.0 mg quantities.

FORMULATION EXAMPLE 9

A subcutaneous formulation may be prepared as follows: IngredientQuantity Active Ingredient 50 mg · mL mg Phosphate buffered saline 1.0ml

FORMULATION EXAMPLE 10

A topical formulation may be prepared as follows: Ingredient QuantityActive Ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g WhiteSoft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

FORMULATION EXAMPLE 11

An intravenous formulation may be prepared as follows: IngredientQuantity Active Ingredient 250 mg Isotonic saline 100 ml

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

Frequently, it will be desirable or necessary to introduce thepharmaceutical composition to the brain, either directly or indirectly.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system used for the transport ofbiological factors to specific anatomical regions of the body isdescribed in U.S. Pat. No. 5,011,472 which is herein incorporated byreference.

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs. Latentiationis generally achieved through blocking of the hydroxy, carbonyl,sulfate, and primary amine groups present on the drug to render the drugmore lipid soluble and amenable to transportation across the blood-brainbarrier. Alternatively, the delivery of hydrophilic drugs may beenhanced by intra-arterial infusion of hypertonic solutions which cantransiently open the blood-brain barrier.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985).

As noted above, the compounds described herein are suitable for use in avariety of drug delivery systems described above. Additionally, in orderto enhance the in vivo serum half-life of the administered compound, thecompounds may be encapsulated, introduced into the lumen of liposomes,prepared as a colloid, or other conventional techniques may be employedwhich provide an extended serum half-life of the compounds. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028 each ofwhich is incorporated herein by reference.

Utility

The conjugates of this invention are alpha4 beta1 (VLA-4) antagonists.Some also have at least a partial affinity for alpha4 beta7 integrins,making them mixed inhibitors of alpha4 integrin. The conjugates provideenhanced in vivo retention as compared to the non-conjugated compounds.The improved retention of the conjugate within the body results in lowerrequired dosages of the drug, which in turn results in fewer sideeffects and reduced likelihood of toxicity. In addition, the drugformulation may be administered less frequently to the patient whileachieving a similar or improved therapeutic effect.

The conjugates of this invention have improved inhibition, in vivo, ofadhesion of leukocytes to endothelial cells mediated by inhibition ofalpha4 beta1 or alpha4 beta7 binding to cellular receptors such asVCAM-1, fibronectin and MadCAM. Preferably, the conjugates of thisinvention can be used, e.g., by infusion, or by subcutaneous injectionor oral administration, for the treatment of diseases mediated by alpha4beta1 and/or alpha4 beta7 or, in general terms, leucocyte adhesion. Theconjugates of the invention can be used to treat a variety ofinflammatory brain disorders, especially central nervous systemdisorders in which the endothelium/leukocyte adhesion mechanism resultsin destruction to otherwise healthy brain tissue. Thus, the conjugatesof the invention can be used for, e.g., the treatment of experimentalautoimmune encephalomyelitis (EAE), multiple sclerosis (MS), meningitis,and encephalitis.

The conjugates of the invention can also be used to treat disorders anddiseases due to tissue damage in other organ systems, i.e., where tissuedamage also occurs via an adhesion mechanism resulting in migration oractivation of leukocytes. Examples of such diseases in mammalianpatients are inflammatory diseases such as asthma, Alzheimer's disease,atherosclerosis, AIDS dementia, diabetes (including acute juvenile onsetdiabetes), inflammatory bowel disease (including ulcerative colitis andCrohn's disease), rheumatoid arthritis, tissue transplantationrejection, tumor metastasis, stroke, and other cerebral traumas,nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemiaand acute leukocyte-mediated lung injury such as that which occurs inadult respiratory distress syndrome.

Still other disease conditions which may be treated using conjugates ofthe invention include erythema nodosum, allergic conjunctivitis, opticneuritis, uveitis, allergic rhinitis, ankylosing spondylitis, psoriaticarthritis, vasculitis, Reiter's syndrome, systemic lupus erythematosus,progressive systemic sclerosis, polymyositis, dermatomyositis, Wegner'sgranulomatosis, aortitis, sarcoidosis, lymphocytopenia, temporalarteritis, pericarditis, myocarditis, congestive heart failure,polyarteritis nodosa, hypersensitivity syndromes, allergy,hypereosinophilic syndromes, Churg-Strauss syndrome, chronic obstructivepulmonary disease, hypersensitivity pneumonitis, chronic activehepatitis, interstitial cystitis, autoimmune endocrine failure, primarybiliary cirrhosis, autoimmune aplastic anemia, chronic persistenthepatitis and thyroiditis.

The invention also provides methods for treating a disease state causedor exacerbated at least in part by alpha 4 integrin-mediated lekocytebinding in a patient, which methods comprise co-administration of aneffective amount of a conjugate of the invention, e.g., a conjugate ofFormula I, and an effective amount of a separate compound which is anα₄β₇ inhibitor. The co-adminstration can be carried out simultaneouslyor sequentially. For example, administration of the conjugate of theinvention can precede adminstration of the α₄β₇ inhibitor by minutes orhours. Alternatively, the α₄β7 inhibitor can be administered prior tothe conjugate of the invention.

Appropriate in vivo models for demonstrating efficacy in treatinginflammatory responses include EAE (experimental autoimmuneencephalomyelitis) in mice, rats, guinea pigs or primates, as well asother inflammatory models dependent upon α₄ integrins.

Inflammatory bowel disease is a collective term for two similar diseasesreferred to as Crohn's disease and ulcerative colitis. Crohn's diseaseis an idiopathic, chronic ulceroconstrictive inflammatory diseasecharacterized by sharply delimited and typically transmural involvementof all layers of the bowel wall by a granulomatous inflammatoryreaction. Any segment of the gastrointestinal tract, from the mouth tothe anus, may be involved, although the disease most commonly affectsthe terminal ileum and/or colon. Ulcerative colitis is an inflammatoryresponse limited largely to the colonic mucosa and submucosa.Lymphocytes and macrophages are numerous in lesions of inflammatorybowel disease and may contribute to inflammatory injury.

Asthma is a disease characterized by increased responsiveness of thetracheobronchial tree to various stimuli potentiating paroxysmalconstriction of the bronchial airways. The stimuli cause release ofvarious mediators of inflammation from IgE-coated mast cells includinghistamine, eosinophilic and neutrophilic chemotactic factors,leukotrines, prostaglandin and platelet activating factor. Release ofthese factors recruits basophils, eosinophils and neutrophils, whichcause inflammatory injury.

Atherosclerosis is a disease of arteries (e.g., coronary, carotid, aortaand iliac). The basic lesion, the atheroma, consists of a raised focalplaque within the intima, having a core of lipid and a covering fibrouscap. Atheromas compromise arterial blood flow and weaken affectedarteries. Myocardial and cerebral infarcts are a major consequence ofthis disease. Macrophages and leukocytes are recruited to atheromas andcontribute to inflammatory injury.

Rheumatoid arthritis is a chronic, relapsing inflammatory disease thatprimarily causes impairment and destruction of joints. Rheumatoidarthritis usually first affects the small joints of the hands and feetbut then may involve the wrists, elbows, ankles and knees. The arthritisresults from interaction of synovial cells with leukocytes thatinfiltrate from the circulation into the synovial lining of the joints.See e.g., Paul, Immunology (3d ed., Raven Press, 1993).

Another indication for the conjugates of this invention is in treatmentof organ or graft rejection mediated by VLA-4. Over recent years therehas been a considerable improvement in the efficiency of surgicaltechniques for transplanting tissues and organs such as skin, kidney,liver, heart, lung, pancreas and bone marrow. Perhaps the principaloutstanding problem is the lack of satisfactory agents for inducingimmunotolerance in the recipient to the transplanted allograft or organ.When allogeneic cells or organs are transplanted into a host (i.e., thedonor and donee are different individuals from the same species), thehost immune system is likely to mount an immune response to foreignantigens in the transplant (host-versus-graft disease) leading todestruction of the transplanted tissue. CD8⁺ cells, CD4 cells andmonocytes are all involved in the rejection of transplant tissues.Conjugates of this invention which bind to alpha4 integrin are useful,inter alia, to block alloantigen-induced immune responses in the doneethereby preventing such cells from participating in the destruction ofthe transplanted tissue or organ. See, e.g., Paul et al., TransplantInternational 9, 420-425 (1996); Georczynski et al., Immunology 87,573-580 (1996); Georcyznski et al., Transplant. Immunol. 3, 55-61(1995); Yang et al., Transplantation 60, 71-76 (1995); Anderson et al.,APMIS 102, 23-27 (1994).

A related use for conjugates of this invention which bind to VLA-4 is inmodulating the immune response involved in “graft versus host” disease(GVHD). See e.g., Schlegel et al., J. Immunol. 155, 3856-3865 (1995).GVHD is a potentially fatal disease that occurs when immunologicallycompetent cells are transferred to an allogeneic recipient. In thissituation, the donor's immunocompetent cells may attack tissues in therecipient. Tissues of the skin, gut epithelia and liver are frequenttargets and may be destroyed during the course of GVHD. The diseasepresents an especially severe problem when immune tissue is beingtransplanted, such as in bone marrow transplantation; but less severeGVHD has also been reported in other cases as well, including heart andliver transplants. The therapeutic agents of the present invention areused, inter alia, to block activation of the donor T-cells therebyinterfering with their ability to lyse target cells in the host.

The formulations of the present invention are especially useful in thetreatment of multiple sclerosis, rheumatoid arthritis and asthma.

A further use of the conjugates of this invention is inhibiting tumormetastasis. Several tumor cells have been reported to express VLA-4 andcompounds which bind VLA-4 block adhesion of such cells to endothelialcells. Steinback et al., Urol. Res. 23,175-83 (1995); Orosz et al., Int.J. Cancer 60, 867-71 (1995); Freedman et al., Leuk. Lymphoma 13, 47-52(1994); Okahara et al., Cancer Res. 54, 3233-6 (1994).

Compounds having the desired biological activity may be modified asnecessary to provide desired properties such as improved pharmacologicalproperties (e.g., in vivo stability, bio-availability), or the abilityto be detected in diagnostic applications. Stability can be assayed in avariety of ways such as by measuring the half-life of the proteinsduring incubation with peptidases or human plasma or serum. A number ofsuch protein stability assays have been described (see, e.g., Verhoef etal., Eur. J. Drug Metab. Pharmacokinet., 1990, 15(2):83-93).

A further use of the conjugates of this invention is in treatingmultiple sclerosis. Multiple sclerosis is a progressive neurologicalautoimmune disease that affects an estimated 250,000 to 350,000 peoplein the United States. Multiple sclerosis is thought to be the result ofa specific autoimmune reaction in which certain leukocytes attack andinitiate the destruction of myelin, the insulating sheath covering nervefibers. In an animal model for multiple sclerosis, murine monoclonalantibodies directed against VLA-4 have been shown to block the adhesionof leukocytes to the endothelium, and thus prevent inflammation of thecentral nervous system and subsequent paralysis in the animals¹⁶.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985).

The amount administered to the patient will vary depending upon what isbeing administered, the purpose of the administration, such asprophylaxis or therapy, the state of the patient, the manner ofadministration, and the like. In therapeutic applications, compositionsare administered to a patient already suffering from a disease in anamount sufficient to cure or at least partially arrest the symptoms ofthe disease and its complications. An amount adequate to accomplish thisis defined as “therapeutically effective dose.” Amounts effective forthis use will depend on the disease condition being treated as well asby the judgment of the attending clinician depending upon factors suchas the severity of the inflammation, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient are in the form ofpharmaceutical compositions described above. These compositions may besterilized by conventional sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration.

The therapeutic dosage of the conjugates of the present invention willvary according to, for example, the particular use for which thetreatment is made, the manner of administration of the conjugate, thehealth and condition of the patient, and the judgment of the prescribingphysician. For example, for intravenous administration, the dose willtypically be in the range of about 20 μg to about 2000 μg per kilogrambody weight, preferably about 20 μg to about 500 μg, more preferablyabout 100 μg to about 300 μg per kilogram body weight. Suitable dosageranges for intranasal administration are generally about 0.1 μg to 1 mgper kilogram body weight. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Conjugates of this invention are also capable of binding or antagonizingthe actions of α₆β₁, α₉β₁, α₄β₇, β_(d)β₂, α_(e)β₇ integrins (althoughα₄β₁ and α₉β₁ are preferred in this invention). Accordingly, conjugatesof this invention are also useful for preventing or reversing thesymptoms, disorders or diseases induced by the binding of theseintegrins to their respective ligands.

For example, International Publication Number WO 98/53817, publishedDec. 3, 1998 (the disclosure of which is incorporated herein byreference in its entirety) and references cited therein describedisorders mediated by α₄β₇. This reference also describes an assay fordetermining antagonism of α₄β₇ dependent binding to VCAM-Ig fusionprotein.

Additionally, compounds that bind α_(d)β₂ and α_(e)β₇ integrins areparticularly useful for the treatment of asthma and related lungdiseases. See, for example, M. H. Grayson et al., J. Exp. Med. 1998,188(11) 2187-2191. Compounds that bind α_(e)β₇ integrin are also usefulfor the treatment of systemic lupus erythematosus (see, for example, M.Pang et al., Arthritis Rheum. 1998, 41(8), 1456-1463); Crohn's disease,ulcerative colitis and inflammatory bowel disease (IBD) (see, forexample, D. Elewaut et al., Scand J. Gastroenterol 1998, 33(7) 743-748);Sjogren's syndrome (see, for example, U. Kroneld et al., Scand J.Gastroenterol 1998, 27(3), 215-218); and rheumatoid arthritis (see, forexample, Scand J. Gastroenterol 1996, 44(3), 293-298). And compoundsthat bind α₆β₁ may be useful in preventing fertilization (see, forexample, H. Chen et al., Chem. Biol. 1999, 6, 1-10).

In another aspect of the invention, the conjugates and compositionsdescribed herein can be used to inhibit immune cell migration from thebloodstream to the central nervous system in the instance of, forexample, multiple sclerosis, or to areas which result ininflammatory-induced destruction of the myelin. Preferably, thesereagents inhibit immune cell migration in a manner that inhibitsdemyelination and that further may promote remyelination. The reagentsmay also prevent demyelination and promote remyelination of the centralnervous system for congenital metabolic disorders in which infiltratingimmune cells affect the development myelin sheath, mainly in the CNS.The reagents preferably also reduce paralysis when administrered to asubject with paralysis induced by a demyelinating disease or condition.

Inflammatory diseases that are included for treatment by thecompositions, conjugates and methods disclosed herein include generallyconditions relating to demyelination. Histologically, myelinabnormalities are either demyelinating or dysmyelinating. Demyelinationimplies the destruction of myelin. Dysmyelination refers to defectiveformation or maintenance of myelin resulting from dysfunction of theoligodendrocytes. Preferably, the compositions and methods disclosedherein are contemplated to treat diseases and conditions relating todemyelination and aid with remyelination. Additional diseases orconditions contemplated for treatment include meningitis, encephalitis,and spinal cord injuries and conditions generally which inducedemyelination as a result of an inflammatory response. The conjugates,compositions and methods disclosed herein are not directed towardsdiseases and conditions wherein there is, for example, a genetic defectleading to improper myelin formation, e.g., dysmyelination.

The compositions, conjugates and cocktails disclosed herein arecontemplated for use in treating conditions and diseases associated withdemyelination. Diseases and conditions involving demyelination include,but are not limited to, multiple sclerosis, congenital metabolicdisorders (e.g., phenylketonuria, Tay-Sachs disease, Niemann-Pickdisease, Gaucher's disease, Hurler's syndrome, Krabbe's disease andother leukodystrophies), neuropathies with abnormal myelination (e.g.,Guillain Barre, chronic immune demyelinating polyneuropathy (CIDP),multifocal CIDP, anti-MAG syndrome, GALOP syndrome, anti-sulfatideantibody syndrome, anti-GM2 antibody syndrome, POEMS syndrome,perineuritis, IgM anti-GD1b antibody syndrome), drug relateddemyelination (e.g., caused by the administration of chloroquine, FK506,perhexiline, procainamide, and zimeldine), other hereditarydemyelinating conditions (e.g., carbohydrate-deficient glycoprotein,Cockayne's syndrome, congenital hypomyelinating, congenital musculardystrophy, Farber's disease, Marinesco-Sjogren syndrome, metachromaticleukodystrophy, Pelizaeus-Merzbacher disease, Refsum disease, prionrelated conditions, and Salla disease) and other demyelinatingconditions (e.g., meningitis, encephalitis or spinal cord injury) ordiseases.

There are various disease models that can be used to study thesediseases in vivo. For example, animal models include but are not limitedto: TABLE III Disease Model Species EAE Mouse, rat, guinea pigMyelin-oligodendrocyte glycoprotein (MOG) Rat induced EAE TNF-αtransgenic model of demyelination MouseMultiple Sclerosis

The most common demyelinating disease is multiple sclerosis, but manyother metabolic and inflammatory disorders result in deficient orabnormal myelination. MS is a chronic neurologic disease, which appearsin early adulthood and progresses to a significant disability in mostcases. There are approximately 350,000 cases of MS in the United Statesalone. Outside of trauma, MS is the most frequent cause of neurologicdisability in early to middle adulthood.

The cause of MS is yet to be determined. MS is characterized by chronicinflammation, demyelination and gliosis (scarring). Demyelination mayresult in either negative or positive effects on axonal conduction.Positive conduction abnormalities include slowed axonal conduction,variable conduction block that occurs in the presence of high-but notlow-frequency trains of impulses or complete conduction block. Positiveconduction abnormalities include ectopic impulse generation,spontaneously or following mechanical stress and abnormal “cross-talk”between demyelinated exons.

T cells reactive against myelin proteins, either myelin basic protein(MBP) or myelin proteolipid protein (PLP) have been observed to mediateCNS inflammation in experimental allergic encephalomyelitis. Patientshave also been observed as having elevated levels of CNS immunoglobulin(Ig). It is further possible that some of the tissue damage observed inMS is mediated by cytokine products of activated T cells, macrophages orastrocytes.

Today, 80% patients diagnosed with MS live 20 years after onset ofillness. Therapies for managing MS include: (1) treatment aimed atmodification of the disease course, including treatment of acuteexacerbation and directed to long-term suppression of the disease; (2)treatment of the symptoms of MS; (3) prevention and treatment of medicalcomplications; and (4) management of secondary personal and socialproblems.

The onset of MS may be dramatic or so mild as to not cause a patient toseek medical attention. The most common symptoms include weakness in oneor more limbs, visual blurring due to optic neuritis, sensorydisturbances, diplopia and ataxia. The course of disease may bestratified into three general categories: (1) relapsing MS, (2) chronicprogressive MS, and (3) inactive MS. Relapsing MS is characterized byrecurrent attacks of neurologic dysfunction. MS attacks generally evolveover days to weeks and may be followed by complete, partial or norecovery. Recovery from attacks generally occurs within weeks to severalmonths from the peak of symptoms, although rarely some recovery maycontinue for 2 or more years.

Chronic progressive MS results in gradually progressive worseningwithout periods of stabilization or remission. This form develops inpatients with a prior history of relapsing MS, although in 20% ofpatients, no relapses can be recalled. Acute relapses also may occurduring the progressive course.

A third form is inactive MS. Inactive MS is characterized by fixedneurologic deficits of variable magnitude. Most patients with inactiveMS have an earlier history of relapsing MS.

Disease course is also dependent on the age of the patient. For example,favourable prognostic factors include early onset (excluding childhood),a relapsing course and little residual disability 5 years after onset.By contrast, poor prognosis is associated with a late age of onset(i.e., age 40 or older) and a progressive course. These variables areinterdependent, since chronic progressive MS tends to begin at a laterage that relapsing MS. Disability from chronic progressive MS is usuallydue to progressive paraplegia or quadriplegia (paralysis) in patients.In one aspect of the invention, patients will preferably be treated whenthe patient is in remission rather then in a relapsing stage of thedisease.

Short-term use of either adrenocorticotropic hormone or oralcorticosteroids (e.g., oral prednisone or intravenousmethylprednisolone) is the only specific therapeutic measure fortreating patients with acute exacerbation of MS.

Newer therapies for MS include treating the patient with interferonbeta-1 b, interferon beta-1a, and Copaxone® (formerly known as copolymer1). These three drugs have been shown to significantly reduce therelapse rate of the disease. These drugs are self-administeredintramuscularly or subcutaneously.

However, none of the current treatment modalities inhibit demyelination,let alone promotes or allows spontaneous remyelination or reducesparalysis. One aspect of the invention contemplates treating MS withagents disclosed herein either alone or in combination with otherstandard treatment modalities.

Congenital Metabolic Disorders

Congenital metabolic disorders include phenylketonuria (PKU) and otheraminoacidurias, Tay-Sachs disease, Niemann-Pick disease, Gaucher'sdisease, Hurler's syndrome, Krabbe's disease and other leukodystrophiesthat impact the developing sheath as described more fully below.

PKU is an inherited error of metabolism caused by a deficiency in theenzyme phenylalanine hydroxylase. Loss of this enzyme results in mentalretardation, organ damage, unusual posture and can, in cases of maternalPKU, severely compromise pregnancy. A model for studying PKU has beendiscovered in mice. Preferably infants identified with PKU are sustainedon a phenylalanine free or lowered diet. An aspect of the inventionwould be to combine such diets with the conjugates and compositionsdisclosed herein to prevent demyelination and remyelinate cells damageddue to PKU.

Classical Tay-Sachs disease appears in the subject at about age 6 monthsand will eventually result in the death of the subject by age 5 years.The disease is due to the lack of the enzyme, hexoaminidase A (hex A),which is necessary for degrading certain fatty substances in the brainand nerve cells. The substances in the absence of the enzyme accumulateand lead to the destruction of nerve cells. Another form of hex A enzymedeficiency occurs later in life and is referred to as juvenile, chronicand adult onset forms of hex A deficiency. Symptoms are similar to thosethat characterize classical Tay-Sachs disease. There is also an adultonset form of the enzyme deficiency. Currently there is no cure ortreatment for the disease/deficiency, only the preventative measure ofin utero testing of the fetus for the disease. Thus, the conjugates andcompositions disclosed herein may be useful in ameliorating orpreventing the destruction of nerve cells in such patients.

Niemann-Pick disease falls into three categories: the acute infantileform, Type B is a less common, chronic, non-neurological form, and TypeC is a biochemically and genetically distinct form of the disease. In anormal individual, cellular cholesterol is imported into lysosomes forprocessing, after which it is released. Cells taken from subjects withNiemann-Pick have been shown to be defective in releasing cholesterolfrom lysosomes. This leads to an excessive build-up of cholesterolinside lysosomes, causing processing errors. NPC1 was found to haveknown sterol-sensing regions similar to those in other proteins, whichsuggests it plays a role in regulating cholesterol traffic. Nosuccessful therapies have been identified for Types A and C forms ofNeumann-Pick. For Type C, patients are recommended to follow alow-cholesterol diet. Thus, the conjugates and compositions disclosedherein may be useful in ameliorating or preventing the destruction ofthe cells.

Gaucher's disease is an inherited illness caused by a gene mutation.Normally, this gene is responsible for an enzyme calledglucocerebrosidase that the body needs to break down the fat,glucocerebroside. In patients with Gaucher's disease, the body is notable to properly produce this enzyme and the fat cannot be broken down.Like Tay-Sachs disease, Gaucher's disease is considerably more common inthe descendants of Jewish people from Eastern Europe (Ashkenazi),although individuals from any ethnic group may be affected. Among theAshkenazi Jewish population, Gaucher's disease is the most commongenetic disorder, with an incidence of approximately 1 in 450 persons.In the general public, Gaucher's disease affects approximately 1 in100,000 persons.

In 1991, enzyme replacement therapy became available as the firsteffective treatment for Gaucher's disease. The treatment consists of amodified form of the glucocerebrosidase enzyme given intravenously. Itis contemplated that the compositions and conjugates disclosed hereincan be used alone or more preferably in combination withglycocerebrosidase administration to treat the disease in an afflictedsubject.

Hurler's syndrome, also known as mucopolysaccharidosis type I, is aclass of overlapping diseases. These genetic diseases share in commonthe cellular accumulation of mucopolysaccharides in fibroblasts. Thediseases are genetically distinguishable. Fibroblast and bone marrowtransplantation does not seem to be helpful, thus conjugates andcompositions useful in ameliorating disease severity and progression areneeded. The conjugates and compositions disclosed herein may beadministered to a subject to ameliorate disease progression and/orseverity.

Krabbe's disease (also known as Globoid cell leukodystrophy) is anautosomal recessive condition resulting from galactosylceramidase (orgalactocerebrosidase) deficiency, a lysosomal enzyme that catabolises amajor lipid component of myelin. Incidence in France is an estimated1:150,000 births. The disease leads to demyelination of the central andperipheral nervous system. Onset generally occurs during the first yearof life and the condition is rapidly progressive, but juvenile,adolescent or adult onset forms have also been reported, with a morevariable rate of progression. Diagnosis is established from enzyme assay(galactosylceramidase deficiency). There are several natural animalmodels (mouse, dog, monkey). Krabbe's disease, like allleukodystrophies, has no known cures or effective treatments. Oneembodiment of the instant invention is to use the compositions andconjugates disclosed herein to treat or ameliorate Krabbe's disease andother leukodystrophies.

Leukodystrophies are a group of genetically determined progressivedisorders that affect the brain, spinal cord and peripheral nerves. Theyinclude adrenoleukodystrophy (ALD), adrenomyeloneuropathy (AMN),Aicardi-Goutiers syndrome, Alexander's disease, CACH (i.e., childhoodataxia with central nervous system hypomyelination or vanishing whitematter disease), CADASIL (i.e., cerebral autosomal dominant arteriopathywith subcortical infarcts and leukoencephalopathy), Canavan disease(spongy degeneration), Cerebrotendinous Xanthomatosis (CTX), Krabbe'sdisease (discussed above), metachromatic leukodystrophy (MLD), neonataladrenoleukodystrophy, ovarioleukodystrophy syndrome,Pelizaeus-Merzbacher disease (X-linked spastic paraglegia), Refsumdisease, van der Knaap syndrome (vaculating leukodystrophy withsubcortical cysts) and Zellweger syndrome. None of the diseases haveeffective treatments let alone cures. Consequently, means of treating orameliorating the symptoms of the disease, such as by using thecompositions and conjugates disclosed herein, is needed.

Neuropathies with Abnormal Myelination

A variety of chronic immune polyneuropathies exist which result indemyelination in the patient. The age of onset for the conditions variesby condition. Standard treatments for these diseases exist and could becombined with the compositions and conjugates disclosed herein.Alternatively, the compositions and conjugates disclosed can be usedalone. Existing standard therapies include the following: TABLE IVNeuropathy Clinical Features Treatment Chronic Immune Onset between 1-80years. T-cell immunosuppression Demyelinating Characterized by weakness,with prednisone, Polyneuropathy (CIDP) sensory loss, and nervecyclosporine A or hypertrophy. methotrexate, HIG, plasma exchangeMultifocal CIDP Onset between 28 to 58 years T cell immunosuppressionand characterized by with prednisone asymmetric weakness, Humanimmunoglobulin (HIG) sensory loss with a course that is slowlyprogressive or relapsing-remitting. Multifocal Motor Onset ranges from25 to 70 HIG Neuropathy (MMN) years, with twice as many B cellimmunosuppression men as women. Features with plasma exchange includeweakness, muscle cyclophosphamide, atrophy, fasciculations, and Rituxancramps which are progressive over 1-30 years. Neuropathy with IgM Onsetis usually over age 50 B-cell immunosuppression binding to Myelin- andis characterized by plasma exchange Associated Glycoprotein sensory loss(100%), cyclophosphamide (MAG) weakness, gain disorder, Rituxan tremorwhich is all slowly α-interferon progressive. cladribine or fludarabineprednisone GALOP Syndrome (Gait A gait disorder with HIG disorderAutoantibody, polyneuropathy Plasma exchange Late-age, Onset,cyclophosphamide Polyneuropathy) POEMS Syndrome Onset occurs between 27and Osteosclerotic lesions are (Polyneuropathy, 80 years with weakness,treated with irradiation. Organomegaly, sensory loss, reduced orWidespread lesions with Endocrinopathy, M- absent tendon reflexes, skinchemotherapy (Melphalan Protein and Skin disorders and other features.and prednisone). changes) also known as Crow-Fukase Syndrome andTakatsuki diseaseDrug and Radiation Induced Demyelination

Certain drugs and radiation can induce demyelination in subjects. Drugsthat are responsible for demyelination include but are not limited tochloroquine, FK506, perhexiline, procainamide, and zimeldine.

Radiation also can induce demyelination. Central nervous system (CNS)toxicity due to radiation is believed to be cause by (1) damage tovessel structures, 10 (2) deletion of oligodendrocyte-2 astrocyteprogenitors and mature oligodendrocytes, (3) deletion of neural stemcell populations in the hippocampus, cerebellum and cortex, andgeneralized alterations of cytokine expression. Most radiation damageresults from radiotherapies administered during the treatment of certaincancers. See for review Belka et al., 2001 Br. J. Cancer 85: 1233-9.However, radiation exposure may also be an issue for astronauts(Hopewell, 1994 Adv. Space Res. 14: 43342) as well as in the event ofexposure to radioactive substances.

Patients who have received drugs or been exposed accidentally orintentionally to radiation may experience a benefit by administered oneof the conjugates or compositions disclosed herein to preventdemyelination or to promote remyelination.

Conditions Involving Demyelination

Additional inherited syndromes/diseases that result in demyelinationinclude Cockayne's syndrome, congenital hypomyelinating, Farber'sdisease, metachromatic leukodystrophy, Peliszaeus-Merzbacher disease,Refsum, prion related conditions and Salla disease.

Cockayne's syndrome (CS) is a rare inherited disorder in which peopleare sensitive to sunlight, have short stature and have the appearance ofpremature aging. In the classical form of Cockayne's syndrome (Type I),the symptoms are progressive and typically become apparent after the ageof one year. An early onset or congenital form of Cockayne's syndrome(Type II) is apparent at birth. Interestingly, unlike other DNA repairdiseases, Cockayne's syndrome is not linked to cancer. CS is amulti-system disorder that causes both profound growth failure of thesoma and brain and progressive cachexia, retinal, cochlear, andneurologic degeneration, with a leukodystrophy and demyelinatingneuropathy without an increase in cancer. After exposure to UV (e.g.,sunlight), subjects with Cockayne's syndrome can no longer performtranscription-coupled repair. Two genes defective in Cockayne'ssyndrome, CSA and CSB, have been identified so far. The CSA gene isfound on chromosome 5. Both genes code for proteins that interacts withcomponents of the transcriptional machinery and with DNA repairproteins.

To date, no cures or effective treatments for patients with this diseasehave been identified. Thus, one aspect of the invention is treatment ofthis disease with the conjugates and compositions disclosed herein.

Congenital hypomyelination has several names including congenitaldysmyelinating neuropathy, congenital hypomyelinating polyneuropathy,congenital hypomyelination (Onion Bulb) polyneuropathy, congenitalhypomyelination neuropathy, congenital neuropathy caused byhypomyelination, hypomyelination neuropathy and CHN. Hereditaryperipheral neuropathies, among the most common genetic disorders inhumans, are a complex, clinically and genetically heterogeneous group ofdisorders that produce progressive deterioration of the peripheralnerves. Congenital hypomyelination is one of a group of disorders. Thisgroup includes hereditary neuropathy with liability to pressure palsies,Charcot-Marie-Tooth disease, Dejerine-Sottas syndrome, and congenitalhypomyelinating neuropathy. There are no known cures or effectivetreatments for any of these disorders.

Farber's disease has several names including: Farber lipogranulomatosis,ceremidase deficiency, acid ceramidase deficiency, AC deficiency,N-laurylsphingosine deacylase deficiency, and N-acylsphingosineamidohydrolase. As certain names reveal, the disease occurs due to adeficiency of acid ceramidase (also known as N-acylsphingosineamidohydrolase, ASAH). The lack of the enzyme results in an accumulationof non-sulfonated acid mucopolysaccharide in the neurons and glialcells. Patients with the disease usually die before the age of 2 years.

Metachromatic leukodystrophy (MLD) is a genetic disorder caused by adeficiency of the enzyme arylsulfatase A. It is one of a group ofgenetic disorders called the leukodystrophies that affect growth of themyelin sheath. There are three forms of MLD: late infantile, juvenile,and adult. In the late infantile form, which is the most common, onsetof symptoms begins between ages 6 months and 2 years. The infant isusually normal at birth, but eventually loses previously gainedabilities. Symptoms include hypotonia (low muscle tone), speechabnormalities, loss of mental abilities, blindness, rigidity (i.e.,uncontrolled muscle tightness), convulsions, impaired swallowing,paralysis, and dementia. Symptoms of the juvenile form begin betweenages 4 and 14, and include impaired school performance, mentaldeterioration, ataxia, seizures, and dementia. In the adult form,symptoms, which begin after age 16, may include impaired concentration,depression, psychiatric disturbances, ataxia, tremor, and dementia.Seizures may occur in the adult form, but are less common than in theother forms. In all three forms mental deterioration is usually thefirst sign.

Peliszaeus-Merzbacher disease (also known as perinatal sudanophilicleukodystrophy) is an X-linked genetic disorder that causes anabnormality of a proteolipid protein. The abnormality results in aninfant's death typically before the age of one year. There are no knowntreatments or cures for the disease.

Refsum disease (also referred to as phytanic acid oxidase deficiency,heredopathia atactica polyneuritiformis or hereditary motor and sensoryneuropathy IV, HMSN IV) is caused by mutations in the gene, whichencodes phytanoyl-CoA hydroxylase (PAHX or PHYH). The major clinicalfeatures are retinitis pigmentosa, chronic polyneuropathy and cerebellarsigns. Phytanic acid, an unusual branched chain fatty acid(3,7,11,15-tetramethyl-hexadecanoic acid) accumulates in the tissues andbody fluids of patients with the disease and is unable to be metaboliseddue to the lack of PAHX. Plasmapheresis performed once or twice monthlyeffectively removes the acid from the body and permits liberalization ofdietary restrictions limiting phytanic acid intake.

Prion related conditions include Gerstmann-Straussler disease (GSD),Creutzfeldt-Jakob disease (CJD), familial fatal insomnia and aberrantisoforms of the prion protein can act as infectious agents in thesedisorders as well as in kuru and scrapie (a disease found in sheep). Theterm prion derives from “protein infectious agent” (Prusiner, Science216: 136-44, 1982). There is a proteolytic cleavage of the prion relatedprotein (PRP) which results in an amyloidogenic peptide that polymerisesinto insoluble fibrils.

Salla disease and other types of sialurias are diseases involvingproblems with sialic acid storage. They are autosomal recessiveneurodegenerative disorders that may present as a severe infantile form(i.e., ISSD) or as a slowly progressive adult form that is prevalent inFinland (i.e., Salla disease). The main symptoms are hypotonia,cerebellar ataxia and mental retardation. These conditions and diseasesare also contemplated for palliative or ameliorating treatments.

Other conditions that result in demyelination include post-infectiousencephalitis (also known as acute disseminated encephalomyelitis, ADEM),meningitis and injuries to the spinal cord. The compositions andconjugates disclosed herein are also contemplated for use in treatingthese other demyelinating conditions.

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention. Unless otherwise stated, alltemperatures are in degrees Celsius.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning.

-   -   ACN=acetonitrile    -   bs=broad singlet    -   d=Doublet    -   dd=doublet of doublets    -   Et₃N=triethylamine    -   g=Grams    -   h and hr=Hour    -   HPLC=High performance (or pressure) liquid chromatography    -   kg=kilogram    -   kDa=kilodalton    -   L=Liter    -   m=multiplet    -   M=Molar    -   mg=milligram    -   min Minute    -   mL=milliliter    -   mm=millimeter    -   mM=millimolar    -   mmol=millimol    -   s=Singlet    -   sat.=saturated    -   t=Triplet    -   TFA=trifluoroacetic acid    -   TLC or tic=thin layer chromatography    -   Ts=Tosyl    -   μL=microliter    -   μg=microgram    -   μm micron or micrometer

General Methods: Proton (¹H) and carbon (¹³C) nuclear magnetic resonancespectra (NMR) were obtained using a Gemini 2000 or Bruker Avance 300spectrometer. The presence of the polyethylene glycol (PEG) protons canbe detected by a large, broad singlet at 3.6 ppm. The integration ofthis signal can vary depending on the size of the PEG moiety. Presenceof the conjugated VLA-4 antagonist can also be detected in the ¹H NMRspectra of conjugates. Thin layer chromatography was performed onpre-coated sheets of silica 60 F₂₅₄ (EMD 15341-1) or pre-coated MKC18Fsilica 60 Å (Whatman 4803-110). Mass spectrometry was performed on anAgilent mass spectrometer (LC/MSD VL) in positive ion single quad mode.

HPLC Methods for PEG Products and PEG Conjugates:

Preparative reverse phase HPLC was performed using a Varian Prep Star(Model SD-1) module with a Varian UV detector set at 210 nm. Method A:Samples of PEG products and PEG conjugates were purified using reversephase HPLC on a Vydac C18, 300 Å pore size column (250 mm×21.2 mm),typically using a gradient of 35-50% ACN+0.1% TFA in 100 min at 20mL/min. Method B: Samples of PEG products and conjugates were purifiedusing reverse phase HPLC on a Vydac C18, 300 Å pore size column (250mm×50 mm), typically using a gradient of 35-50% ACN+0.1% TFA in 100 minat 60 mL/min.

Method C: The purity of PEG products and conjugates was confirmed viareverse phase analytical HPLC using an Agilent Series 1100 Quaternarysystem equipped with a Waters Symmetry 300 A pore size, 3.5μ C18 column(150 mm×4.6 mm), using a gradient of 40-50% ACN w/0.1% TFA at a flowrate of 1.5 mL/min. and coupled to an Agilent 1100 variable wavelengthdetector set at 210 nm and a Sedex 75 evaporative light scatteringdetector (40° C., gain=5)

PEG Reagents: PEG starting materials were acquired through NOFCorporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome, Shibuya-ku,Tokyo 150-6019) or Nektar Therapeutics (150 Industrial Road, San Carlos,Calif. 94070) as follows: 30 kDa PEG diamine (NOF Cat. SunbrightDE-300PA); 5 kDa Boc-NH-PEG-NHS ester (Nektar Cat. 4M530H02); 20 kDatetra-amine (NOF Cat. Sunbright PTE-200PA);

-   -   40 kDa 4-arm PEG alcohol (NOF Cat. Sunbright PTE-40000); 40 kDa        3-arm PEG alcohol (NOF Cat. Sunbright GL-400).

Example 1

Sodium hydroxide (10 g, 0.25 m) is dissolved in water (300 ml). To thissolution 4-nitrophenylalanine (50.3 g, 0.22 m) is added and stirreduntil complete dissolution. To the resulting solution the sodiumcarbonate (28.8 g, 0.26 m) is added and stirred suspension is cooled inan ice bath to +8° C. Benzyl chloroformate (44.7 g, 0.26 m) is addeddropwise with vigorous stirring, maintaining internal temperature in +6°to +9° C. range. The mixture is stirred at +6° C. for additional 1 hr,transferred to the separatory funnel and washed with ether (2×150 ml).Aqueous phase is placed in a large Erlenmeyer flask (2L) and iscautiously acidified with dil. aq. HCl to pH=2 and extracted with ethylacetate (4×500 ml). The combined extracts are washed with water anddried with MgSO4. The solution is filtered and filtrate evaporated,residue is dissolved in ethyl acetate (150 ml) and diluted with hexane(500 ml). Crystalline material is filtered off and rinsed with coldsolvent, air dried to give Cbz-4-nitrophenylalanine, 75 g (99.5% yield).¹H-NMR, DMSO-d₆, (δ): 12.85 (bs, 1H), 8.12 (d, 2H, J=9 Hz), 7.52 (d, 2H,J=9 Hz), 7.30 (m, 5H), 4.95 (s, 2H), 4.28 (m, 1H), 3.32 (bs, 1H), 3.10(m, 2H).¹³C-NMR (8): 173.1, 156.3, 146.6, 137.3, 130.8, 128.5, 128.0,127.8, 123.5, 65.6, 55.1, 36.6. MS (m/z): 367.1 [M+23].

The Cbz-4-nitrophenylalanine (75 g, 0.22 m) is dissolved in dioxane (300ml). The resulted stirred solution is cooled in Dry Ice bath to −20° C.(internal). The liquefied isobutylene (approx. 290 ml) is added followedby conc. sulfuric acid (35 ml) added in three equal portions, 30 minapart. The addition of acid is a very exothermic process, accompanied bysubstantial degree of polymerization. Efficient mechanical stirring isessential at this stage. Resulted mixture is stirred for 20 hr, allowingto warm up to ambient temperature then is cautiously poured into sat.aq. sodium carbonate solution (2L) and diluted with ethyl acetate (600ml). Organic layer is separated and aqueous layer is extracted withethyl acetate (2×200 ml). Combined extracts are washed with water anddried with sodium sulfate. The solution is filtered and evaporated todryness. The residue is taken up in ethyl acetate/hexane mixture (500ml; 1:1) and filtered through plug of silica gel (ca. 2×2 in). Thesilica is rinsed with an additional amount of the same solvent (2 Ltotal) and the filtrates are evaporated to give fully protected4-nitrophenylalanine as a viscous oil, 73 g (83% after two steps).¹H-NMR, CDCl₃, (δ): 8.12 (d, 2H, J=8.4 Hz), 7.36 (m, 7H), 5.35 (m, 1H),5.10 (m, 2H), 4.57 (m, 1H), 3.31 (m, 2H), 1.43 (s, 9H). ¹³C-NMR, CDCl₃,(δ): 169.7, 155.3, 146.9, 143.9, 136.0, 130.2, 128.4, 128.2, 128.0,123.3, 82.9, 66.9, 54.7, 38.2, 31.4, 27.8, 13.9. MS (m/z): 423.1 [M+23].

Protected 4-nitrophenylalanine (73 g, 0.18 m) is dissolved in ethanol(500 ml) and platinum oxide catalyst (1.5 g) is added. The resultingsolution is vigorously stirred in hydrogen atmosphere (50-60 psi) atambient temperature until further hydrogen adsorption ceased (3 hr). Thecatalyst is filtered off and the filtrate is evaporated to dryness, theresidue is taken up in ethyl acetate (200 ml) and filtered through plugof silica gel (2×2 in) using ethyl acetate-hexane mixture (3:2, 2L) torinse silica. The filtrate is concentrated to approx. 200 ml and hexane(500 ml) is added. The crystalline product is filtered off, rinsed withcold solvent and air-dried. Yield—56 g, 84%. ¹H-NMR, CDCl₃, (δ): 7.30(bs, 5H), 6.92 (d, 2H, J=8.1 Hz), 6.58 (d, 2H, J=8.1 Hz), 5.21 (m, 1H),5.10 (d, 2H, J=2.1 Hz), 4.46 (m, 1H), 3.59 (bs, 2H), 2.97 (s, 2H, J=5.4Hz), 1.42 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 170.6, 145.1, 136.3, 130.2,128.3, 127.9, 125.6, 115.0, 81.9, 66.6, 55.2, 37.4, 27.8 MS (m/z): 393.1[M+23].

Example 2

The product of Example 1,4-aminophenylalanine, (20 g, 0.054 m) wasdissolved in ethanol (200 ml) and treated with Hunig's base (21 g, 0.162m, 3 eq) and 2-chloro-3-nitropyridine (10.3 g, 0.65 m, 1.2 eq). Resultedsolution was stirred under nitrogen atmosphere and heated to reflux for24 hr. LC analysis indicated presence of small amount of unreactedamine. The small additional amount of chloronitropyridine (1.1 g, 0.13eq) was added and reflux continued for another 24 hr. Reaction mixturewas cooled and evaporated to dryness. Residue was dissolved in ethylacetate (600 ml) and obtained solution was washed with water (1×200 ml),dil. aq. citric acid (0.2 N, 2×200 ml), brine (1×200 ml) and dried withsodium sulfate. Solids were filtered off and filtrate evaporated to give37 g of deep-red oil, containing expected product contaminated withexcess of chloronitropyridine. Impure product was purified by flashchromatography (Biotage 75L system) eluting with ethyl acetate:hexane(3:17) mixture. Fractions containing pure product were combined andevaporated to give deep-red, viscous oil, 26 g (99%). ¹H-NMR, CDCl₃,(δ): 10.10 (S, 1H), 8.49 (m, 2H), 7.57 (d, 2H, J=9 Hz), 7.35 (bs, 5H),7.19 (d, 2H, J=9 Hz), 6.84 (m, 1H), 5.30 (m, 1H), 5.13 (d, 2H, J=3 Hz),4.57 (m, 1H), 3.11 (m, 2H), 1.45 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 170.4,155.5, 155.1, 150.0, 136.7, 136.3, 135.4, 132.4, 129.9, 128.5, 128.3,128.0, 127.9, 122.2, 113.7, 82.2, 66.7, 55.1, 37.7, 27.8, 20.9. MS(m/z): 493.1 [M+1], 515.1 [M+23].

The red nitro compound (26 g, 0.054 m) was dissolved in THF (350 ml) andplatinum oxide catalyst (1.35 g) was added. Resulted mixture wasvigorously stirred under hydrogen atmosphere (50-60 psi) until hydrogenadsorption ceased (2 hr). Catalyst was filtered off and filtrateevaporated to dryness. Residue was dissolved in ethyl acetate (100 ml)and diluted with hexane (50 ml) till beginning of crystallization.Mixture was further diluted with ethyl acetate/hexane (1:1) mixture (300ml) and was left standing in refrigerator for 3 hr. Crystalline solidswere filtered off, rinsed with cold solvent and air-dried to giveproduct, 23 g, 94%. ¹H-NMR, CDCl₃, (δ): 7.81 (dd, 1H, J1=1.5 Hz, J2=4.8Hz), 7.33 (bs, 5H), 7.17 (d, 2H, J=8.4 Hz), 7.03 (d, 2H, J=8.4 Hz), 6.96(dd, 1H, J1=1.5 Hz, J2=7.5 Hz), 6.75 (dd, 1H, J1=5.0 Hz, J2=7.7 Hz),6.22 (s, 1H), 5.31 (m, 1H), 5.09 (bs, 2H), 4.50 (m, 1H), 3.41 (bs, 2H),3.02 (m, 2H), 1.43 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 170.6, 155.6, 145.5,140.21, 138.8, 136.3, 130.8, 129.9, 128.5, 128.3, 127.9, 123.4, 118.2,117.0, 82.0, 66.6, 55.2, 37.4, 27.9. MS (m/z): 407.1 [M−56], 463.1[M+1], 485.1 [M+23].

The aminopyridine (19 g, 0.041 m) was suspended in dichloromethane (200ml) and CDI (12 g, 0.074 m, 1.8 eq) was added. Resulted mixture wasstirred at ambient temperature for 20 hr. Reaction mixture was washedwith sat. aq. bicarbonate (2×100 ml), brine (1×100 ml) and dried withsodium sulfate. Solids were filtered off and filtrate evaporated todryness. Residue was dissolved in ethyl acetate (hot, 300 ml) and set tocrystallize. Crystalline product was filtered off, rinsed with coldethyl acetate and air-dried to give 19.9 g, 81% of the imidazolone.¹H-NMR, CDCl₃, (δ): 10.63 (s, 1H), 8.06 (d, 1H, J=3 Hz), 7.66 (d, 2H,J=9 Hz), 7.32 (m, 8H), 7.05 (m, 1H), 5.36 (m, 1H), 5.13 (s, 2H), 4.59(m, 1H), 3.17 (m, 2H), 1.45 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 170.4, 155.6,154.3, 143.8, 141.0, 136.2, 135.8, 131.8, 130.2, 128.3, 128.0, 125.9,122.2, 118.3, 116.0, 82.4, 66.8, 55.0, 37.7, 27.8. MS (m/z): 433.1[M−56], 489.2 [M+1], 511.2 [M+23].

Example 3

To a solution of the product of Example 2 (4.0 g, 8.19 mmol) in DMF (40ml) crushed potassium carbonate (1.58 g, 11.47 mmol) was added followedby the addition of methyl bromoacetate (1.0 ml, 11.47 mmol). Thereaction mixture was stirred under nitrogen at room temperature overnight. The reaction mixture was concentrated in vacuo and the residuewas taken up in ethyl acetate (100 ml). The organic phase was washedwith H₂O, brine, dried over Na₂SO₄, filtered, and concentrated in vacuo.The crude material was purified by column chromatography (100% ethylacetate) to yield 4.5 g (100%) of the title compound as a white foam.R_(f)=0.42 (5% MeOH/CH₂Cl₂). MS m/z=561, (M+H)⁺. ¹H NMR (CDCl₃) δ8.10-8.08 (d, 1H), δ 7.67-7.65 (d, 2H), δ 7.37-7.30 (m, 7H), δ 7.20-7.17(m, 1H), δ 7.10-7.05 (m, 1H), δ5.30-5.27 (d, 1H), δ5.11 (s, 2H), δ4.58-4.55 (q, 1H), δ3.81 (s, 3H), δ3.16-3.14 (d, 2H), δ 1.42 (s, 9H).

Example 4

A solution of the product of Example 3 (2.25 g, 4.01 mmol) in MeOH (20ml) with Degussa Pd/C catalyst (113 mgs) was placed under H₂ (55 psi)over night. The reaction mixture was filtered through Celite andconcentrated in vacuo to yield 1.65 g (97%) of the title compound as abrown oil. R_(f)=0.32 (5% MeOH/CH₂Cl₂). MS m/z=449, (M+Na)⁺. ¹H NMR(CDCl₃) δ8.11-8.09 (d, 1H), δ 7.68-7.65 (d, 2H), δ 7.41-7.38 (d, 2H),δ7.20-7.17 (m, 1H), δ7.10-7.06 (m, 1H), δ4.73 (s, 2H), δ3.81 (s, 3H), δ3.67-3.62 (m, 1H), δ 3.16-3.09 (m, 1H), δ2.91-2.84 (m, 1H), δ 1.46 (s,9H).

Example 5

Pyridine-3-sulfonic acid (125 g, 0.78 m) was placed in a 1L, 3-neckedflask equipped with mechanical stirrer, reflux condenser, thermometerand nitrogen inlet. Next, the phosphorus pentachloride (250 g, 1.19 m,1.5 eq) was added, followed immediately by the phosphorus oxychloride(330 ml, 3.8 m, 4.5 eq). The contents of flask were initially stirred atambient temperature for 30 min, then brought slowly to gentle reflux(internal temp. approx. 110° C.) over the next hour, kept at thistemperature for approx. 3.5 hr then allowed over the next 12 hr to coolback to ambient temperature. Gas evolution was observed during thistime. The volatiles were stripped under reduced pressure (at 12 mmHg/40°C.) and yellow semi-solid residue was diluted with DCM (1 L). The slurrywas poured slowly into the stirred, ice-cold sat. aq. bicarbonate,maintaining pH=7. Gas evolution was observed. The organic layer wasseparated and aqueous layer was back-extracted with DCM. The combinedextracts were washed with cold sat. aq. bicarbonate, brine and driedwith magnesium sulfate. The solids were filtered off and filtrateevaporated, leaving pyridine-3-sulfonyl chloride as a pale yellow, oilyliquid, 123 g (93% pure; 88% theory). ¹H-NMR, CDCl₃, (δ): 9.26 (d, 1H),8.98 (dd, 1H), 8.34 (m, 1H), 7.62 (m, 1H). ¹³C-NMR, CDCl₃, (δ): 155.3,147.4, 140.9, 134.6, 124.2.

MS (m/z): 178.0 [M+1].

L-penicillamine (150 g, 1.0 m) was dissolved with stirring in DI water(1500 ml), cooled in ice-bath to +8° C. and treated with formalin (150ml, 37% aq.). The reaction mixture was stirred at +8° C. for 2 hr, thencooling bath was removed and stirring continued for 12 hr. The clearsolution was concentrated under reduced pressure (14 mmHg/50°) leavingwhite residue. The solids were re-suspended, then dissolved in hot MeOH(2500 ml) and left standing at ambient temperature for 12 hr. The white,fluffy precipitate was filtered off and rinsed with cold methanol. Thefiltrate was concentrated and set to crystallize again. The collectedprecipitate was combined with the first crop and dried in vacuum ovenfor 24 hr at 55° C. at 45 mmHg. The yield of(R)-5,5-dimethylthiazolidine-4-carboxylic acid was 138 g (>99% pure; 86%theory). ¹H-NMR, DMSO-d₆, (δ): 4.25 (d, 1H), 4.05 (d, 1H), 3.33 (s, 1H),1.57 (s, 3H), 1.19 (s, 3H). ¹³C-NMR, DMSO-d₆, (6): 170.8, 74.4, 57.6,51.8, 28.9, 27.9. MS (m/z): 162.3 [M+1].

In a 4 L reactor equipped with mechanical stirrer and thermometer, abuffer solution was prepared from potassium monobasic phosphate (43 g,0.31 m) and potassium dibasic phosphate (188.7 g, 1.08 m) in DI water (2L). The (R)-5,5-dimethylthiazolidine-4-carboxylic acid (107 g, 0.675 m)was added and stirred until complete dissolution. The solution wascooled in an ice-bath to +8° C. A separately prepared solution ofpyridine-3-sulfonyl chloride (124 g, 0.695 m) in DCM (125 ml) was addeddropwise to the reactor with vigorous stirring over the 1 hr. The pH ofreaction mixture was monitored and after 4 hr, found to be pH=5 andadjusted to pH=6 by addition of solid bicarbonate. The mixture wasallowed to warm up to ambient temperature over 18 hr. The pH wasadjusted to 2 with dil. aq. sulfuric acid, stirred for 1 hr andprecipitated yellow solids were filtered off, rinsed with water toneutral. The solid cake was transferred into 2 L Erlenmayer flask,suspended in DCM (500 ml) with occasional swirling for 5 min andfiltered off again. The filter cake was washed with DCM and air-dried.The yield of the title compound,(R)-5,5-dimethyl-3-(pyridin-3-ylsulfonyl)thiazolidine-4-carboxylic acidwas 148.9 g (98% pure; 73% theory). ¹H-NMR, DMSO-d₆, (6): 9.05 (d, 1H),8.89 (m, 1H), 8.32 (m, 1H), 7.69 (m, 1H), 4.68 (q, 2H), 4.14 (s, 1H),1.35 (s, 3H), 1.29 (s, 3H). ¹³C-NMR, DMSO-d₆, (6): 170.0, 154.3, 147.9,135.8, 134.1, 124.8, 72.6, 54.3, 50.2, 29.4, 25.0. MS (m/z): 303.2[M+1].

Example 6

To a solution of the product of Example 4 (1.65 g, 3.88 mmol) inacetonitrile (35 ml) was added the product of Example 5 (1.06 g, 3.53mmol), HATU (1.75 g, 3.88 mmol), and triethylamine (5.3 ml). Thehomogeneous brown solution was stirred under nitrogen for 72 hours. Theorganic reaction mixture was concentrated in vacuo, taken up in ethylacetate (40 ml), washed with 1N HCl, sat. NaHCO₃, and brine. The organiclayer was dried over Na₂SO₄, filtered, and concentrated in vacuo toyield 2.67 g (97%) 3 as an orange foam. R_(f)=0.36 (5% MeOH/CH₂Cl₂). MSm/z=711, (M+H)⁺. ¹H NMR (CDCl₃) δ 9.09-9.08 (d, 1H), δ 8.86-8.84 (m,1H), δ 8.18-8.15 (m, 1H), δ 8.07-8.05 (m, 1H), δ 7.66-7.63 (d, 2H), δ7.52-7.48 (m, 1H), δ 7.41-7.38 (d, 2H), δ 7.19-7.16 (m, 1H), δ 7.08-7.04(m, 1H), δ 6.93-6.90 (d, 1H), δ 4.83-4.76 (q, 1H), δ 4.71 (s, 2H), δ4.62-4.59 (d, 1H), δ 4.49-4.46 (d, 1H), δ 3.91 (s, 1H), δ 3.80 (s, 3H),δ 3.22-3.08 (m, 2H), δ 1.46 (s, 9H), δ 1.20-1.17 (d, 6H).

Example 7

To a solution of the product of Example 6 (2.67 g, 3.75 mmol) in THF (12ml) was added a solution of LiOH.H₂O (245 mgs, 5.97 mmol) in H₂O (3 ml).The reaction mixture was stirred at room temperature over night undernitrogen. Upon completion the reaction mixture was concentrated invacuo, dissolved in H₂O (100 ml), and acidified to pH 4 with a 1M HClsolution. The desired product precipitated out as a white solid and wasfiltered and rinsed with H₂O to yield 1.87 g (72%) of the titlecompound. MS m/z=697, (M+H)⁺. ¹H NMR (CD₃OD) δ 9.02 (s, 1H), δ 9.80 (s,1H), δ 8.47-8.44 (d, 1H), δ 8.21-8.19 (d, 1H), δ 7.98-7.96 (d, 1H), δ7.63-7.59 (m, 3H), δ 7.52-7.48 (m, 3H), δ 7.17-7.13 (m, 1H), δ 4.75 (s,2H), δ 4.72-4.61 (m, 3H), δ 4.14 (s, 1H), δ 3.22-3.16 (m, 2H), δ 1.45(s, 9H), 81.25-1.19 (d, 6H). ¹³C NMR (CD₃OD) δ 169.9, 169.5, 168.9,153.1, 152.8, 147.5, 142.8, 140.2, 136.6, 135.8, 134.0, 131.7, 129.9,126.0, 124.2, 123.9, 117.8, 114.9, 81.8, 72.6, 54.1, 49.9, 41.3, 36.4,28.5, 26.6, 23.4.

Example 8

The product of Example 2 (52 g, 0.106 m) was slurried in MeOH (450 ml),hydrogenation catalyst (8.7 g, 5% Pd/C, Degussa) was added and themixture was stirred under the hydrogen atmosphere (60 psi) until furtherabsorption ceased (ca. 2 hrs). THF (150 ml) was added to dissolveprecipitated solids and the solution was filtered through plug ofCelite, using DCM to rinse the filter. The filtrate was evaporated todryness, re-dissolved in DCM (300 ml) and stripped again. This operationwas repeated twice. The foamy solids were kept under high vacuum for 3hrs. The yield of title compound was 38.3 g (101% of theory). ¹H-NMR,CDCl₃, (δ): 8.08 (m, 1H), 7.56 (AB q, 4H), 7.37 (m, 1H), 7.06 (m, 1H),3.68 (m, 1H), 2.03 (m, 2H), 1.49 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 173.8,154.6, 143.9, 141.0, 137.4, 131.5, 130.2, 126.1, 122.3, 118.0, 116.1,81.4, 56.0, 40.6, 27.9. MS (m/z): 299.3 [M−56], 355.4 [M+1], 377.4[M+23].

Example 9

The product of Example 8 (38.3 g, assume 0.106 m) was dissolved in DCM(500 ml) and treated successively with: N-methylmorpholine (27 g, 30 ml,0.266 m; 2.5 eq), HOBt (17.3 g, 0.128 m, 1.2 eq), and the product ofExample 5 (33.8 g, 0.112 m; 1.06 eq). The resulting non-homogenoussolution was cooled in an ice-bath to +4° C. and treated with EDC (22.5g, 0.117 m; 1.1 eq) in one portion. The reaction mixture was stirred,allowing it to warm up to ambient temperature over the next 4 hr andthen for 18 hr more. The solvent was stripped and residue dissolved inethyl acetate (1.2 L), washed with sat. aq. bicarbonate (2×250 ml),water (250 ml), brine (300 ml) and dried with magnesium sulfate. Thesolution was filtered and evaporated to dryness, leaving a light orange,viscous oil, 76 g (>>100%). The crude product was purified by flashchromatography on silica gel (Biotage 75 L, in ethyl-acetate/methanol(3%) mixture. Fractions, containing pure product, were combined andevaporated to give 54 g of of the title compound (yield 83%). ¹H-NMR,CDCl₃, (δ): 10.37 (s, 1H), 9.11 (s, 1H), 8.87 (m, 1H), 8.19 (m, 1H),8.05 (m, 1H), 7.56 (AB q, 4H), 7.52 (m, 1H), 7.36 (m, 1H), 7.06 (m, 2H),4.83 (m, 1H), 4.58 (AB a, 2H), 3.96 (s, 1H), 3.19 (m, 2H), 1.49 (s, 9H),1.22 (s, 3H), 1.18 (s, 3H). ¹³C-NMR, CDCl₃, (δ): 169.7, 167.6, 153.9,148.4, 143.8, 140.9, 135.8, 135.6, 132.9, 131.9, 130.2, 125.9, 123.8,122.1, 118.0, 115.9, 82.8, 73.6, 60.3, 54.8, 53.7, 50.6, 37.8, 29.1,27.8, 23.9, 14.1. MS (m/z): 583.3[M−56], 639.4 [M+1], 661.3 [M+23].

Example 10

To an ice chilled solution of ethyl trifluorobutyrate (15 g, 89 mmol)and ethyl formate (36 mL, 444 mmol) in THF (200 mL) under N₂ was added asolution of 1 M KOtBu in THF (107 mmol, 107 mL) over a 25-minute period.After 15 minutes the ice bath was removed and the reaction mixture wasstirred one hour at room temperature. Additional ethyl formate (18 mL,222 mmol) was then added and the reaction mixture was stirred overnight.The reaction mixture was concentrated and the residue partitionedbetween cold ether (100 mL) and cold water (300 mL). The pH of theaqueous phase was adjusted to 2 with concentrated HCl. The product wasextracted with dichloromethane (1×100 mL, 45×75 mL) and the combinedorganic extracts were washed with brine (1×100 mL), dried (MgSO₄),filtered, and concentrated to yield the title compound as thick oilwhich solidified upon standing, 10.2 g (58.5%). MS (m/z)=198 (M+H)⁺.

Example 11

To a solution of the product of Example 10 (10 g, 51 mmol) anddiethylguanidine sulfate (8.3 g, 25.2 mmol) in EtOH (60 mL) under N₂,was added NaOEt, 21% solution in EtOH (20.7 mL, 55.5 mmol) over a10-minute period. The reaction mixture was then heated at reflux for 5hours. The heterogeneous solution was cooled and poured into cold water(100 mL) to give a homogenous solution. The pH of the solution wasadjusted to approximately 3.5 with conc. HCl and 1 N HCl. A solidprecipitated from solution, which was collected by filtration. The lighttan solid was washed with water and air-dried, yielding 2.9 g, (23%) ofthe title compound. MS (m/z)=250 (M+H)⁺. ¹H NMR (300 MHz, CD₃OD) δ 7.65(br s, 1H), 3.55 (q, 4H), 3.30 (q, 2H), 1.25 (t, 6H).

Example 12

A flask was charged with the product of Example 11 (2.0 g, 8.02 mmol),DIEA (1.5 mL, 8.83 mmol), DMAP (0.98 g, 0.8 mmol), and dichloromethane(30 mL). The mixture was cooled to 0° C. and trifluoroacetic anhydride(1.5 mL, 8.83 mmol) was added. The reaction became homogeneous and wasstirred at 0° C. for 3 hours. The mixture was quenched with sat. NaHCO₃and extracted with dichlorormethane. The organic phase was washed with0.2 N citric acid, dried over Na₂SO₄, filtered, and concentrated invacuo to yield 2.87 g (94%) of the title compound as a brown solid.

¹H NMR (300 MHz, CDCl₃) δ 8.28 (s, 1H), 3.65-3.52 (m, 4H), 3.29-3.19 (q,2H), 1.22-1.17 (t, 6H).

Example 13

A solution of the product of Example 12 (1.3 g, 3.5 mmol),H-Phe(p-NO₂)OtBu (1.1 g, 4.2 mmol), and DIEA (0.9 mL, 5.3 mmol) in CH₃CN(14 mL) under N₂ was heated to reflux overnight. The next day additionalH-Phe(p-NO₂)OtBu (0.8 g, 3 mmol) was added and reflux was continued for3 days. The reaction mixture was then cooled and concentrated Theresidue taken-up in EtOAc (50 mL) and the organic portion washed with0.5 N KHSO₄ (3×50 mL), water (1×50 mL), brine (1×10 mL), dried (MgSO₄),filtered and concentrated to a brownish gum. The crude material waspurified by flash chromatography (5:1 hexanes/EtOAc) to yield 640 mg(38%) of the title compound as a golden gum. TLC: 3:1 hexanes/EtOAc,R_(f)=0.30, MS (m/z)=498 (M+H)⁺, ¹H NMR, (300 MHz, CDCl₃) δ 8.19 (d,2H), 7.80 (s, 1H), 7.25 (d, 2H), 5.19 (br d, 1H), 4.95 (q, 1H),3.70-3.50 (m, 4H), 3.45-3.25 (m, 2H), 3.10 (q, 2H), 1.40 (s, 9H), 1.05(t, 6H).

Example 14

The product of Example 13 (635 mg, 1.27 mmol) was dissolved in absoluteEtOH (5 mL) to which was added 35 mg of Pd/C, 10 wt %. The reaction wassubjected to hydrogenation (45 psi H₂) for 2.5 hours at which time 50mgs of Pd/C, 10 wt % was added and the reaction mixture again subjectedto hydrogenation (45 psi H₂) overnight. The reaction mixture wasfiltered through a pad of Celite and the filtrate was concentrated togive 452 mg (76%) of the title compound. MS (m/z)=468 (M+H)⁺, ¹H NMR(300 MHz, CDCl₃) δ 7.75 (s, 1H), 6.90 (d, 2H), 6.60 (d, 2H), 5.05 (br d,1H), 4.80 (q, 1H), 3.70-3.45 (m, 6H), 3.10-2.90 (m, 4H), 1.40 (s, 9H),1.15 (t, 6H).

Example 15

A solution of the product of Example 14 (598 mg, 1.28 mmol),2-chloro-3-nitropyridine (243 mg, 1.53 mmol), and DIEA (0.67 mL, 3.83mmol) in EtOH (5 mL) under N₂ was heated at reflux. The next day thereaction was cooled and additional 2-chloro-3-nitropyridine (40 mg, 0.25mmol) and DIEA (0.11 mL, 0.60 mmol) was added and the reaction washeated at reflux for one day. The reaction mixture was then concentratedand the residue taken-up in EtOAc (20 mL). The organic phase was washedwith water (2×20 mL). The combined aqueous washes was back extractedwith EtOAc (2×10 mL). The combined organic extracts were washed with 0.2N citric acid (3×20 mL), water (1×10 mL), sat. NaHCO3 (3×20 mL), brine(1×10 mL), dried (MgSO4), filtered and stripped to an orange gum. Thecrude product was purified by flash chromatography eluting with 4:1hexanes/EtOAc (R_(f)=0.14) to yield 610 mg (81%) of the title compoundas a red oil. MS (m/z)=590 (M+H)⁺, ¹H NMR (300 MHz, CDCl₃) δ 10.10 (s,1H), 8.55 (d, 1H), 8.50 (m, 1H), 7.79 (s, 1H), 7.75 (d, 2H), 7.15 (d,2H), 6.80 (q, 1H), 5.10 (br d, 1H), 4.90 (m, 1H), 3.70-3.45 (m, 4H),3.25 (m, 2H), 3.10 (q, 2H), 1.40 (s, 9H), 1.10 (t, 6H).

Example 16

To a solution of the product of Example 15 (610 mg, 1.03 mmol) inabsolute EtOH (5 mL) was added 60 mg of Pd/C, 10 wt %. The mixture wassubjected to hydrogenation (45 psi H₂) overnight. The next day thereaction mix was filtered through Celite and the filtrate concentratedto give 500 mg (87%) of the title compound. MS (m/z)=560 (M+H)⁺, ¹H NMR(300 MHz, CDCl₃) δ 7.85 (d, 2H), 7.80 (s, 1H), 7.20 (d, 2H), 7.05 (d,2H), 7.00 (d, 1H), 7.75 (m, 1H), 6.20 (br s 1H), 5.15 (br s, 1H), 4.85(m, 1H), 3.75-3.45 (m, 4H), 3.40 (br s, 2H), 3.15 (m, 2H), 3.05 (q, 2H),1.40 (s, 9H), 1.15 (t, 6H).

Example 17

A solution of the product of Example 16 (141 mg, 0.250 mmol) and CDI (62mg, 0.378 mmol) in CH₂Cl₂ (3 mL) was stirred overnight. The next dayadditional CDI (30 mg, 0.185 mmol) was added and the reaction wasstirred another day. The reaction mixture was then concentrated andtaken-up in EtOAc (10 mL) and the organic portion washed with 0.2 Ncitric acid (3×5 mL), water (1×5 mL), sat. NaHCO₃ (3×5 mL), brine (1×5mL), dried (MgSO₄), filtered and concentrated to yield 69 mg (47%) thetitle compound as a foam which was used without further purification. MS(m/z)=586 (M+H)⁺, ¹H NMR (300 MHz, CDCl₃) δ 8.20 (br s, 1H), 8.05 (d,1H), 7.80 (s, 1H), 7.65 (d, 2H), 7.90 (m, 3H), 7.05 (m, 1H), 5.15 (br d,1H), 4.95 (m, 1H), 3.70-3.45 (m, 4H), 3.25 (app d, 2H), 3.10 (q, 2H),1.40 (s, 9H), 1.15 (t, 6H).

Example 18

To a solution of 4,6-dichloro-5-aminopyrimidine (5.0 g, 30.7 mmol) inDMSO (30 mL) was added Na₂S.9H₂O (7.4 g, 30.8 mmol). The mixture wasstirred at room temperature overnight. Water (40 mL) was then added tothe mixture and the solution evaporated under reduced pressure toapproximately 6 mL. To this solution was added conc. HCl (0.5 mL) andwater to precipitate the product. The solution was filtered and theorange solid was washed with water and dried to afford 4.3 g (86%) ofthe title compound. ¹H NMR (300 MHz, DMSO-d₆) δ 5.84 (2H, s), 7.79 (1H,s), 14.37 (1H, br s); MS(m/z): MH⁺=162.

Example 19

To the product of Example 18 (4.3 g, 26 mmol) dissolved in conc. NH₄OH(4 mL) was added EtOH (40 mL). To this solution, Raney Nickel (excess)was added in portions. The reaction was stirred at room temperatureovernight and then heated at 80° C. for 2 hrs. The mixture was filteredthrough Celite and the filtrate concentrated. The crude product waspurified by flash chromatography on silica using EtOAc/hexanes to afford1.6 g (47%) of the title compound as a yellow solid. ¹H NMR (300 MHz,DMSO-d₆) δ 5.90 (2H, s), 8.20 (2H, s); MS(m/z) MH⁺=130.

Example 20

To the product of Example 19 (0.51 g, 3.9 mmol) in MeOH (20 mL) and HOAc(0.5 mL) was added CH₃CHO (0.52 mL, 9.2 mmol). Then NaBH₃CN (590 mg, 9.2mmol) was added in one portion. The reaction was stirred at roomtemperature overnight and additional HOAc, CH₃CHO, and NaBH₃CN wereadded. The reaction was stirred overnight, concentrated, and the residuewas taken up in EtOAc and sat. NaHCO₃. The separated aqueous layer wasback extracted with EtOAc. The combined organic layer was dried andconcentrated to a residue. The residue was dissolved in MeOH and treatedwith HOAc, CH₃CHO and NaBH₃CN as described above. Following the work upprocedure described above the crude product was purified by flashchromatography on silica using EtOAc/hexanes, to afford 0.35 g (57%) ofthe title compound as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 1.35 (3H,q, J=12 Hz), 3.29 (2H, m), 4.21 (1H, bs), 8.04 (1H, s), 8.36 (1H, s); MS(m/z): MH⁺158.

Example 21

To the product of Example 20 (70 mg, 0.45 mmol) dissolved in DMF (1 mL)was added TEA (93 uL) and isonicotinoyl chloride (0.12 g, 0.67 mmol).The reaction mixture was stirred at room temperature for 2 days and thenpartitioned between EtOAc and sat. NaHCO₃. The separated aqueous layerwas back extracted with EtOAc. The combined organic layer was dried andconcentrated to give 67 mg (57%) of the title compound which was usedwithout further purification. ¹H NMR (300 MHz, CDCl₃) δ 1.26 (3H),3.65-3.69 (1H), 4.21 (1H), 7.17 (2H), 8.43 (1H), 8.54 (2H), 8.86 (1H)Note: ¹H NMR shows evidence of rotamers as demonstrated of broadness ofall peaks; MS (m/z): MH⁺=263.

Example 22

To a solution of the product of Example 21 (0.11 g, 0.42 mmol) and theproduct of Example 8 (0.135 g, 0.38 mmol) in IPA (2.5 ml) was added DIEA(0.35 ml, 1.9 mmol). The reaction mixture was stirred in a sealed tubeat 130° C. for 2 days. The crude mixture was concentrated and the oilwas purified by flash column chromatography with a solvent gradient of0-10% MeOH in CH₂Cl₂ to yield the title compound as an oil. ¹H NMR (300MHz, CDCl₃) δ 1.16 (1.2H, m), 1.26-1.31 (1.8H, m), 1.50-1.53 (9H, d, J=9Hz), 3.0 (1H, m), 3.2 (0.8H, m), 3.36 (1.2H, m), 4.12-4.18 (1.2H, m),4.96-5.10 (0.8H, m), 5.80-5.95 (1H, m), 6.93-6.96 (1H, m), 7.07 (1H, m),7.31-7.45 (5H, m), 7.66-7.75 (3H, m), 8.06 (1H, m), 8.44-8.51 (2H, m);HPLC/MS: single peak at 1.29 min, MH⁺=581.

Example 23

To 2,4-dichloro-5-nitropyrimidine (2.0 g, 10.3 mmol) in MeOH (7 mL) at0° C. under N₂ was added NaOMe (0.5 M in MeOH, 25 mL) dropwise. Afterthe addition was completed, the reaction mixture was stirred at 0 C for15 min. Then diethylamine (5 mL) was added and the mixture was stirredat rt overnight. The reaction mixture was concentrated and the residuewas partitioned between EtOAc and H₂O. The organic layer was dried andconcentrated to a residue which was purified by flash chromatography onsilica using EtOAc/Hexanes, to afford the title compouns as an off whitesolid (1.1 g, 4.9 mmol, 47% yield). ¹H NMR (300 MHz, CDCl3) δ 1.26 (6H,t, J=6.6 Hz), 3.70 (4H, m), 4.08 (3H, s), 9.01 (1H, s); HPLC/MS:MH⁺=227.

Example 24

To the product of Example 23 (1.1 g, 4.9 mmol) in MeOH/EtOAc (1:1, 20mL) was reduced with Pd/C (5% degussa, 0.5 g) and H₂ (50 psi) in a Parrshaker overnight. The reaction mixture was filtered and the filtratedwas concentrated under reduced pressure to afford the title compound asa solid (0.85 g, 4.3 mmol, 88.5% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.18(6H, t, J=6.9 Hz), 3.03 (2H, br), 3.57 (6H, t, J=6.9 Hz), 3.96 (3H, s),7.71 (1H, s); HPLC/MS: MH⁺=197.

Example 25

To the product of Example 24 (0.85 g, 4.3 mmol) in CH₂Cl₂ (15 mL) andTEA (1.4 mL, 10 mmol) was added isonicotinyl chloride HCl salt (1.139,6.3 mmol). After 15 min, TLC showed no starting material. The mixturewas extracted between EtOAc and sat. NaHCO₃. The aqueous layer waswashed with EtOAc twice. The combined organic layers were washed withsat. NaHCO₃ and brine. It was dried over MgSO₄ and filtered. Thefiltrate was concentrated to give the title compound as a brown solid(1.3 g, 4.3 mmol, 100% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.20 (6H, t,J=6.9 Hz), 3.60 (4H, q, J=6.9 Hz), 3.96 (3H, s), 7.72 (2H, d, J=6.0 Hz),7.75 (1H, bs), 8.80 (2H, d, J=6.0 Hz), 8.89 (1H, s); HPLC/MS: MH⁺=302.

Example 26

To the product of Example 25 (100 mg, 0.33 mmol) in THF (1 mL) was addedKOtBu (1M in THF, 0.5 mL) slowly followed by Etl (40 μL, 0.5 mmol). Thereaction mixture was stirred at rt overnight. TLC showed thedisappearance of the starting material. The mixture was partitionedbetween EtOAc and H₂O. The aqueous layer was washed with EtOAc. Thecombined organic layers were washed with sat. NaHCO₃ and brine. It wasdried and concentrated to give the title compound (90 mg, 0.27 mmol,83%) that was used without further purification. ¹H NMR (300 MHz, CDCl₃)δ 1.10 (9H, m), 3.47 (5H, m), 3.92 (1H, m), 7.14 (2H, d, J=6.0 Hz), 7.78(1H, bs), 8.44 (2H, d, J=6.0 Hz); HPLC/MS: MH⁺=330.

Example 27

To the product of Example 26 (200 mg, 0.61 mmol) in DMF (4 mL) was addedEtSNa (66 mg, 0.79 mmol) and the reaction mixture was heated at 100 Cfor 1 hr. LC/MS showed starting material still present. Another portionof NaSEt (66 mg, 0.79 mmol) was added and the reaction heated foranother 2 hr. LC/MS showed product only. DMF was removed under reducedpressure and H₂O (10 mL) was added followed by conc. HCl (0.132 mL).Evaporating of the solvent left a residue. It was dissolved in EtOH andfiltered. The filtrate was concentrated to to yield the title compound(190 mg, 100%) that was used without further purification. ¹H NMR (300MHz, CD₃OD) δ 1.24 (9H, m), 3.60 (4H, m), 3.60-4.00 (2H, br), 8.12 (3H,d, J=5.7 Hz), 8.92 (2H, d, J=5.7 Hz); HPLC/MS: MH⁺=316.

Example 28

To the product of Example 27 (70 mg, 0.22 mmol) in POCl₃ (3 mL) at rtwas added diethylaniline (30 μL). The reaction mixture was heated to 100C for 30 min. Then it was concentrated. The residue was partitionedbetween EtOAc and H₂O. The organic layer was washed with H₂O twice. Thenit was dried and concentrated to give the title compound (50 mg, 0.15mmol, 68%) and used for the next reaction without further purification.HPLC/MS: MH⁺=334

Example 29

To a solution of the product of Example 28 (50 mg, 0.15 mmol) and theproduct of Example 8 (60 mg, 0.17 mmol) in IPA (0.75 mL) was added DIEA(0.15 mL, 0.8 mmol). The reaction mixture was stirred in a sealed tubeat 130 degrees for 7 days. The crude mixture was concentrated and theresidue was purified by preparative HPLC and silica gel flashchromatography to yield an off white solid (10 mg). ¹H NMR (300 MHz,CDCl3) δ 1.10-1.30 (9H, m), 1.48 (4.5H, s), 1.51 (4.5H, s), 2.80-3.38(3H, m), 3.53 (4H, m), 4.054.30 (1H, m), 4.83 (0.5H, m), 4.96 (0.5H, m),5.15-5.50 (1H, m), 6.95-7.10 (2H, m), 7.25-7.50 (5H, m), 7.69 (0.5H, d,J=8.4 Hz), 7.76 (0.5H, d, J=8.4 Hz), 8.08 (1H, d, J=5.1 Hz), 8.51 (2H,m), 8.83 (0.5H, br), 8.95 (0.5H, br);

HPLC/MS: MH⁺=652.

Example 30

Compound 25 (20 g, 0.11 mol) was dissolved in CH₂Cl₂ (500 mL) under N₂.The reaction mixture was cooled to 0° C. Triethylamine (18.12 mL, 0.13mol) was added, followed by trifluoroacetic anhydride (18.14 mL, 0.13mol) in portions. The reaction was allowed to warm to room temperatureovernight. The reaction mixture was concentrated in vacuo and theresidue was taken up in ethyl acetate (200 mL). The organic phase waswashed with H₂O, sat. NaHCO₃, brine, dried over Na₂SO₄, filtered, andconcentrated in vacuo to yield 29.7 g (96%) 29 as a yellow solid. ¹H NMR(CDCl₃) δ 3.64-3.60 (m, 2H), 3.55-3.53 (m, 2H), 3.49-3.45 (m, 4H), 1.44(s, 9H). ¹³C NMR (CDCl₃) δ 155.7 (J_(C-F)=36 Hz), 154.3, 116.4(J_(C-F)=288 Hz), 80.8, 45.7, 43.3, 28.3.

Compound 29 (29.26 g, 0.10 mol) was added in portions to a 500 mL flaskcontaining a solution of 4N HCL in dioxane (200 mL) at 0° C. Thereaction was stirred in ice bath for 4 hours when TLC (3:1 hexanes:ethylacetate) showed 100% conversion to product. The reaction mixture wasconcentrated in vacuo and treated with ethyl ether (500 mL). The productwas filtered and dried to yield 22.5 g (99%) 30 as a whitemono-hydrochloride salt. ¹H NMR (DMSO-d₆) δ 3.82-3.79 (m, 4H), 3.53 (s,1H), 3.18-3.16 (m, 4H). ¹³C NMR (DMSO-d₆) δ 154.3 (J_(C-F)=35 Hz), 115.9(J_(C-F)=289 Hz), 66.1, 42.0, 41.9, 41.5.

A 250 mL flask was charged with 30 (1.0 g, 4.6 mmol), CH₂Cl₂ (40 mL),and sat. NaHCO₃ (40 mL). The reaction mixture was stirred vigorously at0° C. for 15 minutes. Stirring was ceased and the layers were allowed toseparate. A 2.0 M solution of phosgene in toluene (9 mL, 18 mmol) wasadded to the reaction mixture which was stirred vigorously for 30minutes, maintaining temperature at 0° C. The layers were separated andthe aqueous phase was washed with CH₂Cl₂ (15 mL). The combined organiclayers were washed with brine, dried over Na₂SO₄, filtered, andconcentrated in vacuo. The residue was taken up in CH₂Cl₂ andconcentrated in vacuo again to yield 1.0 g (92%) 31 as a white solid. MS(m/z) 245, (M+H)⁺. ¹H NMR (CDCl₃) δ 3.80-3.68 (m, 8H). ¹³C NMR (CDCl₃) δ155.9 (J_(C-F)=37 Hz), 148.7 (J_(C-F)=12 Hz), 116.3 (J_(C-F)=289 Hz),48.3, 47.8, 45.7, 45.3, 45.1, 42.9, 42.7.

A 25 mL flask was charged with 24 (5.97 g, 0.011 mol), DMAP (1.34 g,0.011 mol), and CH₂Cl₂ (22 mL). Triethylamine (2.4 mL, 0.017 mol) wasadded followed by 31 (4.2 g, 0.017 mol). The reaction mixture was heatedat reflux for 20 hours. The reaction mixture was concentrated in vacuoand the residue was taken up in ethyl acetate. The organic phase waswashed with sat. NaHCO₃, H₂O, brine, dried over Na₂SO₄, filtered, andconcentrated in vacuo to yield 9.3 g pink foam. The crude material waspurified by flash chromatography (gradient of 50% ethyl acetate/hexanesto 75% ethyl acetate/hexanes) to yield 6.1 g (76%) 32 as a pale pinkfoam. R_(f)=0.14 (1:1 hexanes:ethyl acetate). MS (m/z) 730, (M+H)⁺. ¹HNMR (CDCl₃) δ 9.08-9.07 (m, 1H), 8.87-8.85 (m, 1H), 8.16-8.14 (m, 1H),7.52-7.48 (m, 1H), 7.25-7.22 (d, 2H), 7.03-7.00 (d, 2H), 6.91-6.88 (d,1H), 4.784.70 (q, 1H), 4.60-4.44 (dd, 2H), 3.88 (s, 1H), 3.75-3.60 (m,8H), 3.09-3.06 (m, 2H), 1.42 (s, 9H), 1.18 (s, 3H), 1.16 (s, 3H).

To a solution of 32 (6.11 g, 8.4 mmol) dissolved in MeOH (90 mL) wasadded a solution of potassium carbonate (5.79 g, 42 mmol) in H₂O (10mL). The reaction was stirred at room temperature for 15 minutes andthen concentrated in vacuo. The residue was filtered and washed withcopious amounts of H₂O to yield 4.65 g (88%) 33 as a white solid.R_(f)=0.08 (5% MeOH/CH₂Cl₂). MS (m/z) 634, (M+H)⁺. ¹H NMR (CDCl₃) δ9.09-9.08 (m, 1H), 8.87-8.85 (m, 1H), 8.16-8.14 (m, 1H), 7.52-7.48 (m,1H), 7.23-7.20 (d, 2H), 7.03-7.00 (d, 2H), 6.91-6.88 (d, 1H), 4.78-4.70(q, 1H), 4.59-4.46 (dd, 2H), 3.89 (s, 1H), 3.65-3.50 (m, 4H), 3.09-3.06(m, 2H), 2.92-2.88 (m, 4H), 1.43 (s, 9H), 1.19 (s, 3H), 1.17 (s, 3H).¹³C NMR (CDCl₃) δ 170.1, 167.9, 154.5, 153.9, 150.7, 148.8, 136.0,133.4, 133.2, 130.6, 124.1, 121.9, 83.0, 73.9, 55.0, 53.7, 50.7, 46.0,45.7, 45.0, 37.9, 29.3, 28.0, 24.0.

A 250 mL flask was charged with 33 (2.5 g, 3.9 mmol), CH₂Cl₂ (40 mL),and sat. NaHCO₃ (40 mL). The reaction mixture was stirred vigorously at0° C. for 15 minutes. Stirring was ceased and the layers were allowed toseparate. A 2.0 M solution of phosgene in toluene (7.9 mL, 16 mmol) wasquickly added to the reaction mixture, which was stirred vigorously for60 minutes maintaining the temperature at 0° C. The layers wereseparated and the aqueous phase was washed with CH₂Cl₂ (30 mL). Thecombined organic layers were washed with 0.2 N citric acid, brine, driedover Na₂SO₄, filtered, and concentrated in vacuo to yield 2.8 g (100%)white foam. The crude material was purified through a silica plug,eluting with 100% ethyl acetate, to yield 2.2 g (78%) 40 as a whitefoam. R_(f)=0.43 (3:1 ethyl acetate:hexanes). ¹H NMR (CDCl₃) δ 9.09-9.08(m, 1H), 8.87-8.85 (m, 1H), 8.16-8.14 (d, 1H), 7.52-7.48 (m, 1H),7.25-7.22 (d, 2H), 7.03-7.01 (d, 2H), 6.90-6.88 (d, 1H), 4.78-4.70 (q,1H), 4.60-4.45 (dd, 2H), 3.88 (s, 1H), 3.79-3.65 (m, 8H), 3.10-3.07 (m,2H), 1.43 (s, 9H), 1.18 (s, 3H), 1.17 (s, 3H). ¹³C NMR (CDCl₃) δ 169.9,167.9, 154.1, 153.6, 150.2, 148.5, 136.1, 133.8, 130.6, 124.2, 121.7,82.9, 73.7, 54.8, 53.8, 50.6, 48.3, 45.8, 37.7, 29.2, 27.9, 23.9.

Example 31

A. Synthesis of Carbamate-Linked bis-PEG Conjugate t-butyl Ester

The carbamate linked conjugates were prepared based on a method modifiedfrom WO 92/16555, which is hereby incorporated by reference. Thus, the 6kDa PEG-diol (500 mg, 0.083 mmol) was dissolved in a minimal amount ofCH₂Cl₂(0.1 mL). To this was added a 2.0 M solution of phosgene intoluene (0.6 mL, 1.2 mmol). The reaction mixture was stirred at roomtemperature for 18 hours and then concentrated in vacuo to yield 500 mg(100%) of the 6 kDa PEG-diol (500 mg, 0.083 mmol) was dissolved in aminimal amount of Ch₂Cl₂ (0.1 mL). To this was added a 2.0 M solution ofphosgene in toluene (0.6 mL, 1.2 mmol). The reaction mixture was stirredat room temperature for 18 hours and then concentrated in vacuo to yield500 mg (100%) of the 6 kDa PEG bis-chloroformate as a white solid.

A solution of 33 (211 mg, 0.33 mmol) in CH₂Cl₂ (3 mL)(see example 30)was added to the 6 kDa PEG bid-chloroformate (500 mg, 0.08 mmol)dissolved in CH₂Cl₂(2 mL). Triethylamine (11 μL, 0.08 mmol) was added,and the reaction mixture was stirred at room temperature for 18 hours.The reaction mixture was vacuo, and the residue was dissolved in MeOH(10 mL). 2% cross-linked polystyrene sulfonic acid resin (410 mg) wasadded, and the reaction vessel was swirled for 2 hours. The mixture wasfiltered, and the filtrate was concentrated in vacuo to yield 500 mg(87%) of a white solid. A portion of the material (246 mg) was purifiedby HPLC, yielding 156 mg of the 6 kDa PEG bis-conjugate t-butyl ester asa white solid. HPLC determined the conjugate to be >99% pure (retentiontime=9.655 min).

¹H NMR (CDCl₃) δ 9.07 (bs, 2H), 8.86-8.84 (m, 2H), 8.18-8.15 (d, 2H),7.53-7.48 (m, 2H), 7.22-7.19 (d, 4H), 7.03-6.99 (d, 4H), 6.86-6.83 (d,2H), 4.73-4.70 (m, 2H) 4.58-4.44 (dd, 4H), 4.27-4.24 (m, 4H), 3.62 (bs,621H), 3.40-3.37 (m, 6H), 3.07-3.05 (m, 4H), 1.41 (s, 18H), 1.20-1.16(d, 12H).

B. Synthesis of Carbamate-Linked bis-PEG Conjugate

The purified 6 kDa carbamate-linked bis-PEG conjugate t-butyl ester (100mg, 0.01 mmol) was dissolved in formic acid (5 mL) and heated at 40° C.for 24 hours. The reaction was concentrated in vacuo. The residue wasdissolved in water, concentrated in vacuo, dissolved again in water, andlyophilized to yield 100 mg (100%) of the 6 kDa carbamate-linked bis-PEGconjugate carboxylic acid as a white powder. HPLC determined conjugateto be >99% pure (retention time=7.63 min).

¹H NMR (CDCl₃) δ 9.06 (bs, 2H), 8.84-8.83 (m, 2H), 8.17-8.14 (d, 2H),7.53-7.49 (m, 2H), 7.24-7.21 (d, 4H), 7.02-6.99 (d, 4H), 6.94-6.92 (d,2H), 4.81-4.79 (m, 2H), 4.57-4.48 (dd, 4H), 4.28-4.25 (m, 4H) 3.64 (bs,621H), 3.41-3.38 (m, 6H), 3.23-3.08 (m, 4H), 1.23-1.18 (d, 12H).

Example 32

A. Synthesis of Carbamate-Linked Octa-PEG Conjugate t-butyl Ester

By following the procedures used in Example 31 above and employing anocta-pegylated hub molecule, the title compound was prepared.

Example 33

Nitro-phenyl Ester (101)

A solution of 100 (100 mg, 0.14 mmol) and 4-nitrophenol (24 mg, 0.17mmol) in THF (0.7 mL) was cooled in an ice bath. A suspension of EDC (33mg, 0.17 mmol) in CH₂Cl₂ (0.7 mL) was added and the reaction was stirredat 0° C. for 4 hours. The reaction was diluted with ethyl acetate (100mL) and washed with 0.2 N citric acid. The organic layer was washed with10% K₂CO₃, brine, dried over Na₂SO₄, filtered, and concentrated in vacuoto yield 90 mg (96%) of 101, which was used immediately. ¹H NMR (CDCl₃)δ 9.07 (bs, 1H), 8.84-8.83 (d, 1H), 8.28-8.25 (d, 2H), 8.16-8.14 (d,1H), 8.09-8.07 (d, 1H), 7.65-7.63 (d, 2H), 7.51-7.47 (dd, 1H), 7.41-7.39(d, 2H), 7.36-7.35 (d, 2H), 7.12-7.07 (m, 1H), 6.95-6.92 (d, 1H), 5.00(s, 2H), 4.824.76 (m, 1H), 4.62-4.45 (dd, 2H), 3.91 (s, 1H), 3.18-3.12(m, 2H), 1.44 (s, 9H), 1.18-1.16 (d, 6H).

40 kDa Boc-Protected PEG Diamine

The 30 kDa PEG diamine (1 g, 0.033 mmol) and the 5 kDa Boc-NH-PEG-NHSester (0.67 g, 0.13 mmol) were dissolved in CH₂Cl₂ (10 mL).Diisopropylethylamine (0.116 mL, 0.67 mmol) was added and the reactionstirred at room temperature for 18 hours. The reaction was concentratedin vacuo to yield crude product. The residue was purified according toHPLC Method B to yield 0.46 g of the 40 kDa Boc-protected PEG diamine asa white solid. HPLC Method C determined the product to be >96% pure(retention time=7.6 minutes). ¹H NMR (CDCl₃) δ 6.75 (bs, 2H), 5.15 (bs,2H) 3.64 (s, 2940H, PEG), 3.33-3.31 (m, 10H), 2.47-2.43 (m, 4H), 1.44(s, 18H).

40 kDa PEG Diamine

The 40 kDa Boc-protected PEG diamine (0.2 g, 0.005 mmol) was dissolvedin TFA (4 mL) and stirred at room temperature for 2 hours. The reactionwas concentrated in vacuo to yield 200 mg (100%) crude 40 kDa PEGdiamine as a beige residue. HPLC Method C determined the product tobe >96% pure (retention time=6.5 minutes). ¹H NMR (CDCl₃) δ 7.85 (bs,1H), 6.75 (bs, 1H), 3.64 (s, 2432H, PEG), 3.34-3.32 (m, 10H), 2.47-2.45(m, 4H).

t-butyl Ester (102)

The 40 kDa PEG diamine (0.2 g, 0.005 mmol) was dissolved in CH₂Cl₂ (4mL). Diisopropylethylamine (17 μL, 0.1 mmol) was added, followed bycompound 101 (0.082 g, 0.1 mmol). Another portion ofdiisopropylethylamine (17 μL) was added and the reaction was stirred atroom temperature for 18 hours. The reaction was concentrated in vacuo toyield 300 mg (150%) crude 102 as a white solid. HPLC Method C determinedthe product to be >70% pure (retention time=8.9 minutes). Crude productwas used as is.

Conjugate 103

102 (0.3 g, 0.007 mmol) was dissolved in formic acid (5 mL) and heatedat 40° C. for 24 hours. The reaction was concentrated in vacuo andpurified according to HPLC Method A to yield 0.14 g (68%) of 103 as awhite solid. HPLC Method C determined the conjugate to be >99% pure(retention time=7.3 minutes). ¹H NMR (CDCl₃) δ 9.05 (bs, 2H), 8.82-8.81(m, 2H), 8.17-8.14 (d, 2H), 8.05-8.04 (d, 2H), 7.65-7.58 (m, 4H),7.54-7.48 (m, 2H), 7.41-7.34 (d, 4H), 7.10-7.05 (m, 2H) 6.95-6.93 (d,2H), 4.90 (m, 2H), 4.63-4.49 (m, 6H), 3.64 (bs, 3042H, PEG), 3.35-3.29(m, 6H), 3.22 (m, 5H), 2.45-2.41 (t, 4H), 1.79-1.74 (m, 4H), 1.29-1.27(d, 12H).

Example 34

Synthesis of Polymer:

40 kDa Boc-Protected PEG Tetra-amine

The 20 kDa PEG tetra-amine (0.5 g, 0.025 mmol) and the 5 kDaBoc-NH-PEG-NHS ester (1 g, 0.2 mmol) were dissolved in CH₂Cl₂ (5 mL).Diisopropylethylamine (0.087 mL, 0.5 mmol) was added and the reactionstirred at room temperature for 18 hours. The reaction was concentratedin vacuo and taken up in MeOH (10 mL). 2% cross-linked polystyrenesulfonic acid resin (1.17 g) was added and the reaction vessel wasswirled for 2 hours. The mixture was filtered and concentrated in vacuoto yield 1.4 g crude product as a beige solid. The residue was purifiedaccording to HPLC Method B to yield 0.44 g (44%) of the 40 kDaBoc-protected PEG tetra-amine as a white solid. HPLC Method C determinedthe product to be >96% pure (retention time=8.4 minutes). ¹H NMR (CDCl₃)δ 6.75 (bs, 1H), 5.15 (bs, 1H), 3.64 (s, 2970H, PEG), 3.33-3.29 (m,15H), 2.46-2.42 (t, 8H), 1.79-1.75 (m, 8H), 1.44 (s, 36H).

40 kDa PEG Tetra-amine

The 40 kDa Boc-protected PEG tetra-amine (0.1 g, 0.0025 mmol) wasdissolved in TFA (4 mL) and stirred at room temperature for 1.5 hours.The reaction was concentrated in vacuo to yield 120 mg 40 kDa PEGtetra-amine as a transparent residue. HPLC Method C determined theproduct to be >96% pure (retention time=6.2 minutes). ¹H NMR (CDCl₃) δ7.39 (bs, 1H), 6.75 (bs, 1H), 4.49-4.48 (m, 4H), 3.64 (s, 3253H, PEG),3.35-3.33 (m, 15H), 2.49-2.46 (m, 8H), 1.80-1.75 (m, 8H).

t-butyl Ester (104)

The 40 kDa PEG tetra-amine (0.1 g, 0.0025 mmol) was dissolved in CH₂Cl₂(2 mL). Diisopropylethylamine (9 μL, 0.05 mmol) was added, followed bycompound 101 (82 mg, 0.1 mmol). Another portion of diisopropylethylamine(9 μL) was added and the reaction was stirred at room temperature for 48hours. The reaction was concentrated in vacuo to yield 110 mg crude 104as a white solid. HPLC Method C determined the product to be >80% pure(retention time=10.9 minutes).

Conjugate 105

104 (0.1 g, 0.0024 mmol) was dissolved in formic acid (5 mL) and heatedat 40° C. for 24 hours. The reaction was concentrated in vacuo and waspurified according to HPLC Method A to yield 0.05 g (48%) of 105 as awhite solid. HPLC Method C determined the conjugate to be >99% pure(retention time=7.6 minutes). ¹H NMR (CDCl₃) δ 9.06 (bs, 4H), 8.83-8.82(m, 4H), 8.20-8.17 (d, 4H), 8.05-8.03 (d, 4H), 7.63-7.61 (m, 8H),7.53-7.49 (m, 4H), 7.42-7.33 (m, 8H), 7.09-7.05 (m, 4H) 6.70 (m, 4H),4.84 (m, 4H), 4.62-4.50 (m, 12H), 3.64 (bs, 2357H, PEG), 3.36-3.29 (m,12H), 2.46-2.42 (t, 8H), 1.79-1.74 (m, 8H), 1.30-1.25 (m, 24H).

Example 35

t-butyl Ester (106)

The 40 kDa PEG tetra-amine (37 mg, 0.000925 mmol) and DMAP (0.5 mg,0.0037 mmol) were dissolved in CH₂Cl₂ (0.5 mL). Triethylamine (3 μL,0.019 mmol) was added, followed by 40 (26 mg, 0.037 mmol). Anotherportion of triethylamine (3 μL) was added and the reaction was stirredat room temperature for 18 hours. The reaction was concentrated in vacuoto yield 34 mg crude 106 as a white solid. HPLC Method C determined theproduct to be >80% pure (retention time=10.9 minutes).

Conjugate 107

106 (34 mg, 0.0008 mmol) was dissolved in formic acid (4 mL) and heatedat 40° C. for 24 hours. The reaction was concentrated in vacuo andpurified according to HPLC Method A to yield 17 mg (50%) of 107 as awhite solid. HPLC Method C determined the conjugate to be >99% pure(retention time=7.6 minutes). ¹H NMR (CDCl₃) δ9.06 (bs, 4H), 8.86 (bs,4H), 8.17-8.15 (d, 4H), 7.52 (d, 4H), 7.26-7.23 (d, 8H), 7.02-6.99 (d,8H), 6.72 (m, 4H), 5.69 (m, 4H), 4.80 (m, 4H), 4.60-4.47 (dd, 8H), 3.64(bs, 1602H, PEG), 3.36-3.30 (dd, 8H), 3.16 (m, 8H), 2.46-2.42 (t, 8H),1.24 (bs 24H).

Example 36

40 kDa PEG Tetra-chloroformate

The 40 kDa 4-arm PEG alcohol (0.2 g, 0.005 mmol) was dissolved in CH₂Cl₂(1 mL). To this was added a 2.0 M solution of phosgene in toluene (0.15mL, 0.3 mmol). The reaction was stirred at room temperature for 18hours. The reaction was concentrated in vacuo to yield 200 mg of the 40kDa PEG tetra-chloroformate as a white solid.

t-butyl Ester (108)

The 40 kDa PEG tetra-chloroformate (0.2 g, 0.005 mmol) was dissolved inCH₂Cl₂ (2 mL). To this was added 33 (63 mg, 0.1 mmol), followed bytriethylamine (3.5 μL, 0.025 mmol). The reaction was stirred at roomtemperature for 72 hours. The reaction was concentrated in vacuo toyield 270 mg of 108 as a white solid.

Conjugate 109

108 (0.26 g, 0.006 mmol) was dissolved in formic acid (5 mL) and heatedat 40° C. for 24 hours. The reaction was concentrated in vacuo and waspurified according to HPLC Method A to yield 0.105 g (42%) of 109 as awhite solid. HPLC Method C determined the conjugate to be >99% pure(retention time=8.3 minutes). ¹H NMR (CDCl₃) δ 9.06 (bs, 4H), 8.85-8.84(m, 4H), 8.17-8.14 (d, 4H), 7.53-7.49 (m, 4H), 7.26-7.22 (d, 8H),7.01-6.98 (d, 8H), 4.814.78 (m, 4H), 4.594.46 (dd, 8H), 4.284.35 (m,8H), 3.64 (bs, 3872H, PEG), 3.15-3.13 (m, 8H), 1.24-1.19 (m, 24H).

Example 37

t-butyl Ester (111)

The 40 kDa 3-arm PEG alcohol (0.25 g, 0.00625 mmol), 110 (0.04 g, 0.056mmol), and triphenylphosphine (0.025 g, 0.094 mmol) were dried byazeotropic distillation from toluene (5 mL). Half of the volume wasdistilled over (2.5 mL), and the mixture was cooled to room temperature.CH₂Cl₂ (0.5 mL) was added to make the reaction homogeneous.Diethylazodicarboxylate (0.015 mL, 0.094 mmol) was added drop-wise andthe reaction stirred for 48 hours. HPLC Method C showed the completedisappearance of the starting PEG alcohol. The reaction was concentratedin vacuo to yield the t-butyl ester 111 as a white solid.

Conjugate 112

111 (0.2 g, 0.005 mmol) was dissolved in formic acid (3 mL) and heatedat 40° C. for 24 hours. The reaction was concentrated in vacuo and waspurified according to HPLC Method A to yield 0.1 g (48%) of 112 as awhite solid. HPLC Method C determined the conjugate to be >99% pure(retention time=8.1 minutes). ¹H NMR (CDCl₃) δ 9.08 (bs, 3H), 8.84 (bs,3H), 8.18-8.16 (d, 3H), 8.02-8.00 (d, 3H), 7.67-7.61 (m, 6H), 7.47-7.38(m, 9H), 7.08-7.04 (m, 3H), 6.91 (m, 3H), 4.88 (m, 3H), 4.62-4.49 (dd,6H), 4.13 (m, 6H), 3.64 (bs, 5919H PEG), 3.23 (m, 6H), 1.25-1.24 (d,18H).

Similar methods were used to synthesize the following conjugates:

Example 38

40 kDa 4-arm PEG alcohol was coupled to 110 and deprotected to finalproduct using similar methods as with 112. The product was purifiedaccording to HPLC Method A. HPLC Method C determined the conjugate tobe >95% pure (retention time=7.5-8.1 minutes). ¹H NMR (CDCl₃) δ 9.08(bs, 4H), 8.84 (bs, 4H), 8.18-8.16 (d, 4H), 8.02-8.00 (d, 4H), 7.67-7.61(m, 8H), 7.47-7.38 (m, 12H), 7.08-7.04 (m, 4H), 6.91 (m, 4H), 4.88 (m,4H), 4.62-4.49 (dd, 8H), 4.13 (m, 8H), 3.64 (bs, 10101H PEG), 3.23 (m,8H), 1.25-1.24 (d, 24H).

Example 39

40 kDa 3-arm PEG alcohol was coupled to the t-butyl ester 114 (shownbelow) and deprotected to final product using similar methods as 112.The product was purified according to HPLC Method A. HPLC Method Cdetermined the conjugate to be >95% pure (retention time=7.3 minutes).¹H NMR (CDCl₃) δ 8.66 (bs, 3H), 8.44 (bs, 3H), 8.04-8.02 (d, 3H),7.75-7.30 (m, 24H), 7.10-7.06 (m, 3H), 6.93 (s, 3H), 5.60-5.50 (m, 3H),4.15 (m, 6H), 3.66 (bs, 4270H PEG), 3.00 (m, 3H), 3.40-3.20 (m, 6H),1.27 (d, 9H).

Example 40

t-butyl Ester (117)

The 40 kDa 3-arm PEG alcohol (0.00625 mmol), 116 (0.056 mmol), andtriphenylphosphine (0.094 mmol) are dried by azeotropic distillationfrom toluene (5 mL). Half of the volume is distilled over (2.5 mL), andthe mixture is cooled to room temperature. CH₂Cl₂ (0.5 mL) is added tomake the reaction homogeneous. Diethylazodicarboxylate (0.094 mmol) isadded drop-wise and the reaction stirred for 48 hours. The reaction isconcentrated in vacuo to yield the t-butyl ester 111.

Conjugate 118

118 (0.005 mmol) is dissolved in formic acid (3 mL) and heated at 40° C.for 24 hours. The reaction is concentrated in vacuo and is purifiedaccording to HPLC Method A to yield 112.

Example 41

Using the product of Example 29 and the PEG polymers used in Examples 38and 39, the following conjugates are prepared:

The following conjugates in Tables V and VI are prepared according tothe examples and schemes described herein. TABLE V

Comp. No. t B Moieties 1 2 —C(O)O(CH₂CH₂O)_(p)—C(O)— homo dimer 2 3homot rimer

3 4 homot etram er

4 8 Z homo octom er 5 2 —C(O)O(CH₂CH₂O)_(p)—C(O)— homo dimer 6 homotrimer

7 homot etram erc

8 homo Z octom er 9 homo —C(O)O(CH₂CH₂O)_(p)—C(O)— dimer 10  homo—C(O)O(CH₂CH₂O)_(p)—C(O)— dimer Comp. No. A Moieties 1

2

3

4

5

6

7

8

9

10 First A Moiety

Second A Moiety

Z =

where in each of the structures the sum of all p's is from 200 to 1360.TABLE VI

B Moieties A Moieties \ZZ/(total Mw of conjugate is about/42,000)

\ZZ/(total Mw of conjugate is about/42,000)

\ZZ/(total Mw of conjugate is about/41,000)

(total Mw of conjugate is about 41,000)

(total Mw of conjugate is about 41,500)

(total Mw of conjugate is about 42,000)

(total Mw of conjugate is about 41,500) ZZ =

ZZZ =

BIOLOGICAL EXAMPLES Example A

In Vitro Assays for Determining Potency of Candidate Compounds: 15/7Epitope Induction on Jurkat™ (15/7 LIBS)

Log-phase Jurkat™ cells are incubated in a 96 wells Flexiplate under thefollowing conditions: 10⁵ cells/100 μl/well in assay buffer (20 mMHepes, 140 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂ and 0.3% BSA), 10 μg/ml 15/7(Elan), and compound at a range of concentrations. The incubation isperformed for 30 minutes at RT. Cells are then washed twice with assaybuffer and incubated with Goat Fab′2 anti Ms IgG (Fc)-PE (Immunotechcat# PN IM0551) at 1:200 in assay buffer for 30 minutes on ice in thedark. Cells are then washed once and re-suspended in 300 μl cold assaybuffer for FACS analysis (Becton-Dickinson).

Multivalent Ligand Competition Assay (MVCOMP)

Methods:

Integrin receptors mediate cell adhesion through a multivalentinteraction with their specific ligands—multiple integrin receptorssimultaneously engaging multiple ligand molecules within an adhesivesubstrate. To simulate this physiological interaction in a highlysensitive, quantitative assay, a multivalent ligand probe was developedthat binds specifically to a4 integrin on the surface of lymphocytes.The probe consists of a small molecule ligand for a4 integrin, compound200 (structure shown below), conjugated to a mouse IgG carrier moleculein a 6-10-fold molar excess (6-10 small molecule: 1 IgG). This bindingis inhibited by 21/6, and antibody to a4. Once bound to the cellsurface, the conjugate can be measured with a fluorescently-labeledsecondary antibody against mouse IgG by FACS analysis. In this assay twodifferent mouse monoclonal IgG carrier molecules have been used, TM2aand 27/1, neither of which bind to human lymphocytes unless conjugatedwith the a4 ligand.

Small Molecule-Antibody Reagent Conjugation

Approximately 1 mg of TM2a or 27/1 (Elan) antibody was incubated with a1:6 or 1:10 molar excess of compound 200 in the presence of[Bis(sulfosuccinimidyl)suberate] (Pierce) at 50-fold molar excess in a1.0 ml total volume for 60 minutes at room temperature, with stirring.The reaction was then quenched with the amine-containing buffer TRIS-CIat pH 7.5 for 20 minutes. The product was dialyzed twice for 24 hoursagainst 4,000 volumes PBS at 4 degrees C. in 10 KD MW cutoff membranecassettes to remove unbound small molecule and linking reagent.

Competitive Binding Assay

Jurkat cells (subline™, subclone #15, Elan) were incubated with atitration of various test compounds in the presence of the TM2a or 27/1conjugate diluted at 1:100 in assay buffer for 30 minutes at roomtemperature. Unbound reagent was then removed by several wash steps inwhich the cells were pelleted in a Beckman table top centrifuge at 300×gfor 5 minutes, then resuspended in fresh buffer. Remaining boundantibody conjugate was detected by incubating the cells with GoatF(ab)′₂ anti-mouse IgG(Fc)-Phycoerythrin (BeckmanCoulter) for 30-minutesat 4 degrees C., followed by washing and FACS analysis.

2G3 Epitope Induction on 8866 Cells (2G3 Ligand Induced Binding Site)

Log-phase 8866 cells are incubated in a 96 wells Flexiplate under thefollowing conditions: 10⁵ cells/100 μl/well in assay buffer (PBS, 1 mMCaCl₂, 1 mM MgCl₂, 5% FBS), 10 μg/ml 2G3 (Elan), and compound at a rangeof concentrations. The incubation is performed for 30 minutes at RT.Cells are then washed twice with assay buffer and incubated with GoatFab′₂ anti Ms IgG (Fc)-PE (Immunotech cat# PN IM0551) at 1:200 in assaybuffer for 30 minutes on ice in the dark. Cells are then washed once andre-suspended in 300 μl cold assay buffer for FACS analysis(Becton-Dickinson).

1. A conjugate of the formula I:

B is a bio-compatible polymer moiety optionally covalently attached to abranched-arm hub molecule; q is from about 2 to about 100; A at eachoccurrence is independently a compound of formula II

or a pharmaceutically acceptable salt thereof, wherein J is selectedfrom: a) a group of formula (a):

wherein R³¹ is a covalent bond to the polymer moiety which optionallycomprises a linker, or R³¹ is selected from the group consisting of —H,R^(3′), —NH₂, —NH R^(31′), —N(R^(31′))₂, —NC₃—C₆cyclic, —OR^(31′), and—SR³¹′, wherein each R^(31′) is independently an optionally substitutedstraight or branched C₁-C₆alkyl, optionally substituted C₃-C₆cycloalkyl,optionally substituted aryl, or optionally substituted heteroaryl, andR³² is a covalent bond to the polymer moiety which optionally comprisesa linker, or R³² is selected from the group consisting of —H, —NO₂,haloalkyl, and —N(MR⁴¹)R⁴² wherein M is a covalent bond, —C(O)— or—SO₂—, and R⁴¹ is R^(4′), N(R^(4′))₂, OR^(41′), wherein each R^(41′) isindependently hydrogen, an optionally substituted straight or branchedC₁-C₆alkyl, optionally substituted cycloalkyl, optionally substitutedaryl, optionally substituted heterocyclic or an optionally substitutedheteroaryl, wherein optional substitutions are halide, C₁-C₆alkyl, or—OC₁-C₆alkyl, and R⁴² is hydrogen, R^(41′), alkynyl, or substitutedalkynyl; and b) a group of formula (b):

wherein R is selected from the group consisting of a hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; Ar¹ is selected from the group consisting of aryl,substituted aryl, heteroaryl and substituted heteroaryl wherein each ofaryl, substituted aryl, heteroaryl and substituted heteroaryl isoptionally covalently bound to the polymer moiety wherein the polymermoiety optionally comprises a linker which covalently links the polymermoiety to Ar¹; Ar² is selected from the group consisting of aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substitutedalkyl, alkylamino, and substituted alkylamino, wherein Ar² is optionallycovalently bound to the polymer moiety and wherein the polymer moietyoptionally comprises a linker which covalently links the polymer moietyto Ar²; X is selected from the group consisting of —NR¹—, —O—, —S—,—SO—, —SO₂ and optionally substituted —CH₂— which is optionallycovalently bound to the polymer moiety wherein, in each case, thepolymer moiety optionally comprises a linker which covalently links thepolymer moiety; where R¹ is selected from the group consisting ofhydrogen and alkyl; T is selected from: a) a group of formula (c)

wherein Y is selected from the group consisting of —O— and —NR¹— whereinR¹ is selected from the group consisting of hydrogen and alkyl; W isselected from the group consisting of a covalent bond to a polymermoiety which optionally comprises a linker and —NR²R³ wherein R² and R³are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, and where R² and R³, together with the nitrogen atombound thereto, form a heterocyclic ring or a substituted heterocyclicring wherein each of alkyl, substituted alkyl, heterocyclic andsubstituted heterocyclic is optionally covalently bound to a polymermoiety which further optionally comprises a linker; m is an integerequal to 0, 1 or 2; n is an integer equal to 0, 1 or 2; and b) a groupof formula (d)

wherein G is an optionally substituted aryl or optionally substitutedheteroaryl 5 or 6 membered ring containing 0 to 3 nitrogens, whereinsaid aryl or heteroary optionally further comprises a covalent bond to apolymer moiety which optionally comprises a linker; R⁶ is a covalentbond to a polymer moiety which optionally comprises a linker, or R⁶ is—H, alkyl, substituted alkyl, or —CH₂C(O)R¹⁷, wherein R¹⁷ is —OH, —OR¹⁸,or —NHR¹⁸, wherein R¹⁸ is alkyl, substituted alkyl, aryl, substitutedaryl, heteroaryl, or substituted heteroaryl; R⁵⁵ is —OH or ahydrolyzable ester, or R⁵⁵ forms a hydrolyzable polymer ester with thepolymer moiety, optionally through a linker; provided that: A. at leastone of J, R⁵⁵, and T contains a covalent bond to the polymer moiety; B.when X is —O—, then m is two; and C. the conjugate of formula I has amolecular weight of no more than about 80,000.
 2. The conjugateaccording to claim 1, wherein only one of J, R⁵⁵ and T contains acovalent bond to a polymer moiety.
 3. The conjugate according to claim1, wherein n is 2, R at each occurrence is C₁-C₃ alkyl, and both Rgroups are on the same carbon.
 4. The conjugate according to claim 1,wherein, q is an integer of from 2 to about
 20. 5. The conjugateaccording to claim 1, wherein q is an integer of from 2 to about
 8. 6.The conjugate according to claim 1, wherein A at each occurrence isindependently a compound of formula IIa

or a pharmaceutically acceptable salt thereof.
 7. The conjugateaccording to claim 1, wherein A at each occurrence is independently acompound of formula IIb:

or a pharmaceutically acceptable salt thereof.
 8. The conjugateaccording to claim 1, wherein A at each occurrence is independently acompound of IIc:

or a pharmaceutically acceptable salt thereof.
 9. The conjugateaccording to claim 1, wherein A at each occurrence is independently acompound of formula IId:

or a pharmaceutically acceptable salt thereof.
 10. The conjugateaccording to claim 1, wherein A at each occurrence is independently acompound of formula IIe:

or a pharmaceutically acceptable salt thereof.
 11. The conjugateaccording to claim 1, wherein A at each occurrence is independently acompound of formula IIf:

or a pharmaceutically acceptable salt thereof, wherein R⁴ is covalentlybound to a polymer moiety which optionally comprises a linker; R⁵ isselected from the group consisting of alkyl and substituted alkyl; andAr³ is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl.
 12. The conjugate according toclaim 1, wherein A at each occurrence is independently a compound of

or a pharmaceutically acceptable salt thereof. R⁴ is covalently bound toa polymer moiety which optionally comprises a linker; R⁵ is selectedfrom the group consisting of alkyl and substituted alkyl; and Ar³ isselected from the group consisting of aryl, substituted aryl, heteroaryland substituted heteroaryl; and n is an integer equal to 0, 1 or
 2. 13.The conjugate according to claim 1, wherein A at each occurrence isindependently a compound of formula IIh below:

or a pharmaceutically acceptable salt thereof, wherein R⁴ is covalentlybound to a polymer moiety which optionally comprises a linker; and Ar³is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl.
 14. The conjugate according toclaim 1, wherein A at each occurrence is independently a compound offormula IIi:

or a pharmaceutically acceptable salt thereof.
 15. The conjugateaccording to claim 14 wherein m is 1, X is S, and R at each occurrrenceis independently selected from hydroxyl, alkyloxy, alkyl, or a covalentbond to the polymer moiety.
 16. The conjugate according to claim 15wherein n is 2, and R at both occurrences is methyl.
 17. The conjugateaccording to claim 1, wherein A at each occurrence is independently acompound of formula IIj below:

or a pharmaceutically acceptable salt thereof.
 18. The conjugateaccording to claim 1 wherein A at each occurrence is independently acompound of formula IIk below:

or a pharmaceutically acceptable salt thereof.
 19. The conjugateaccording to claim 1 wherein A at each occurrence is independently acompound of formula IIL below:

or a pharmaceutically acceptable salt thereof, wherein R⁴ is covalentlybound to the polymer moiety which optionally comprises a linker.
 20. Theconjugate according to claim 17 wherein G is pyridinyl, R31 is hydrogenor dialkylamino, and R32 is sulfonamide, amide, or urea.
 21. Theconjugate according to claim 1, wherein A and B are as shown below: BMoieties —C(O)O(CH₂CH₂O)_(p)—C(O)—

Z —C(O)O(CH₂CH₂O)_(p)—C(O)—

Z —C(O)O(CH₂CH₂O)_(p)—C(O)— ZZ (total Mw of conjugate is about 42,000)ZZ (total Mw of conjugate is about 42,000) ZZZ (total Mw of conjugate isabout 41,000)

(total Mw of conjugate is about 42,000)

(total Mw of conjugate is about 41,500)

(total Mw of conjugate is about 42,000)

(total Mw of conjugate is about 41,500)

(total Mw of conjugate is about 42,000)

(total Mw of conjugate is about 41,500) A Moieties

wherein Z is

ZZ is

ZZZ is

where the sum of all p's is from 100 to
 1360. 22. A conjugate accordingto claim 1, selected from the group consisting of:

and a pharmaceutically acceptable salt thereof.
 23. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of a conjugate according to claim 1 ormixtures thereof.
 24. A pharmaceutical composition according to claim 23wherein the pharmaceutically acceptable carrier is suitable forparenteral administration.
 25. A pharmaceutical composition according toclaim 23 wherein the pharmaceutically acceptable carrier is suitable forsubcutaneous administration.
 26. A pharmaceutical composition accordingto claim 23 wherein the pharmaceutically acceptable carrier is suitablefor administration by infusion.
 27. A pharmaceutical compositionaccording to claim 23 wherein the pharmaceutically acceptable carrier issuitable for administration by injection.
 28. A pharmaceuticalcomposition according to claim 23 wherein the pharmaceuticallyacceptable carrier is suitable for oral administration.
 29. Apharmaceutical composition according to claim 23 wherein thepharmaceutically acceptable carrier is suitable for rectaladministration.
 30. A pharmaceutical composition according to claim 23wherein the pharmaceutically acceptable carrier is suitable foradministration using a patch.
 31. A pharmaceutical composition accordingto claim 23 wherein the pharmaceutically acceptable carrier is suitablefor administration by inhalation.
 32. A method for treating a diseasestate caused or exacerbated at least in part by alpha 4integrin-mediated leukocyte binding in a patient, which method comprisesadministering an effective amount of a conjugate according to claim 1.33. The method of claim 32 wherein the a4-binding interaction that isinhibited is with VCAM-1.
 34. The method of claim 32 wherein the a4binding interaction that is inhibited is with fibronectin.
 35. Themethod of claim 32 wherein the a4 binding interaction that is inhbitiedis with MadCAM.
 36. The method according to claim 32 wherein saiddisease state is an autoimmune disease state.
 37. The method of claim 36wherein treatment by the conjugate of claim 1 alleviates theinflammation and subsequent tissue damage caused by an autoimmunereaction.
 38. The method of claim 32 wherein the disease state ismultiple sclerosis, meningitis, encephalitis, stroke, and other cerebraltraumas
 39. The method of claim 32 wherein the disease state is multiplesclerosis.
 40. The method according to claim 32 wherein said diseasestate is selected from the group consisting of asthma, adult respiratorydistress syndrome and acute leukocyte-mediated lung injury.
 41. Themethod according to claim 40 wherein the disease state is asthma. 42.The method according to claim 32 wherein the disease condition isrheumatoid arthritis.
 43. The method according to claim 32 wherein thedisease state is an inflammatory disease condition selected from thegroup consisting of erythema nodosum, allergic conjunctivitis, opticneuritis, uveitis, allergic rhinitis, ankylosing spondylitis, psoriaticarthritis, vasculitis, Reiter's syndrome, systemic lupus erythematosus,progressive systemic sclerosis, polymyositis, dermatomyositis, Wegner'sgranulomatosis, aortitis, sarcoidosis, lymphocytopenia, temporalarteritis, pericarditis, myocarditis, congestive heart failure,polyarteritis nodosa, hypersensitivity syndromes, allergy,hypereosinophilic syndromes, Churg-Strauss syndrome, chronic obstructivepulmonary disease, hypersensitivity pneumonitis, chronic activehepatitis, interstitial cystitis, autoimmune endocrine failure, primarybiliary cirrhosis, autoimmune aplastic anemia, chronic persistenthepatitis and thyroiditis, AIDS dementia, diabetes, Alzheimer's disease,dementia, atherosclerosis, tumor metastasis, and transplant rejection.44. The method of claim 32 wherein the disease state is Sjogren'sdisease.
 45. The method of claim 32 wherein the disease state is Crohn'sdisease.
 46. The method of claim 32, wherein the disease state isinflammatory bowel disease.
 47. The method of claim 32, wherein thedisease state is ulcerative colitis.
 48. The method of claim 32, whereinthe conjugate is an α₄β₁ and an α₄β₇ inhibitor.
 49. A pharmaceuticalcomposition comprising a conjugate according to claim 1 in combinationwith an α₄β₇ inhibitor.
 50. A method for treating a disease state causedor exacerbated at least in part by alpha 4 integrin-mediated leukocytebinding in a patient, which method comprises co-administration of aneffective amount of a conjugate according to claim 1 and an effectiveamount of an α₄β₇ inhibitor.